Inkjet deposition of reagents for histological samples

ABSTRACT

Devices and methods for the deposition of reagents onto cells or tissue samples are disclosed. Also disclosed are reagent compositions suitable for dispensing via a droplet-on-demand system.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of International PatentApplication No. PCT/EP2016/058801 filed Apr. 20, 2016, which claimspriority to and the benefit of U.S. Provisional Patent Application No.62/150,122 filed Apr. 20, 2015. Each of these related patentapplications is incorporated by reference herein.

BACKGROUND OF THE DISCLOSURE

Molecular pathology is the examination at a molecular level of the DNA,mRNA, and proteins that cause or are otherwise associated with disease.From this examination important information about patient diagnosis,prognosis, and treatment options can be elucidated. Diseases, such ascancer, can be diagnosed by a number of different methods. One method isto identify the presence of a biomarker, such as a cancer biomarker, intissue or cells, the biomarker being correlated, or thought to becorrelated, with a particular cancer type.

Hematoxylin and eosin (H&E) are primary stains that have been used forat least a century and are essential for recognizing various tissuetypes and the morphologic changes that form the basis of contemporarycancer diagnosis. The stain works well with a variety of fixatives anddisplays a broad range of cytoplasmic, nuclear, and extracellular matrixfeatures. Hematoxylin has a deep blue-purple color and stains nucleicacids by a complex reaction. Eosin is pink and stains proteinsnonspecifically. In a typical tissue, nuclei are stained blue, whereasthe cytoplasm and extracellular matrix have varying degrees of pinkstaining. Well-fixed cells show considerable intranuclear detail. Nucleishow varying cell-type- and cancer-type-specific patterns ofcondensation of heterochromatin (hematoxylin staining) that arediagnostically very important. Nucleoli stain with eosin. If abundantpolyribosomes are present, the cytoplasm will have a distinct blue cast.The Golgi zone can be tentatively identified by the absence of stainingin a region next to the nucleus. Thus, the stain discloses abundantstructural information, with specific functional implications.

Histochemistry and cytochemistry are techniques often used to identifybiomarkers within the context of intact cells by labeling the sampleswith molecules that bind specifically to the biomarker in a manner thatcan be visualized on a microscope. Immunohistochemistry (IHC) andimmunocytochemistry (ICC) are types of histochemistry and cytochemistrythat use antibodies to label the biomarkers. By identifying thebiomarker in the context of a tissue environment or cellularenvironment, spatial relationships between the biomarkers and othermorphological or molecular features of the cell or tissue sample can beelucidated, which may reveal information that is not apparent from othermolecular or cellular techniques.

These techniques typically require a series of treatment steps conductedon a tissue section (e.g. a tumor biopsy) or cell sample (e.g. blood orbone marrow) mounted on a microscope slide, such as a glass, plastic, orquartz microscope slide. Frequently used steps include pretreatments toprepare the tissue samples for mounting and staining (e.g.deparaffinization, rehydration, and/or and antigen retrieval), labelingof the tissue sample with biomarker-specific antibody or probe, enzymelabeled secondary treatment and incubation, substrate reaction with theenzyme to produce a fluorophore or chromophore highlighting areas of thesample labeled for the biomarker, counterstaining, and the like. Most ofthese steps are separated by multiple rinse steps to remove unreactedresidual reagent from the prior step. Incubations frequently areconducted at elevated temperatures, usually around 37° C., and thetissue must be continuously protected from dehydration.

In view of the large number of repetitive treatment steps needed forIHC, automated systems have been introduced to reduce human labor andthe costs and error rate associated therewith, and to introduceuniformity. Examples of automated systems that have been successfullyemployed include the ES®, NexES®, DISCOVERY™, BENCHMARK™ and Gen II®staining systems available from Ventana Medical Systems (Tucson, Ariz.).These systems employ a microprocessor controlled system including arevolving carousel supporting radially positioned slides. A steppermotor rotates the carousel placing each slide under one of a series ofreagent dispensers positioned above the slides. Bar codes on the slidesand reagent dispensers permits the computer controlled positioning ofthe dispensers and slides so that different reagent treatments can beperformed for each of the various tissue samples by appropriateprogramming of the computer.

In order to introduce reagents and other fluids during processing, areagent delivery system and method is often used. Typically, the regentdelivery system automatically pipettes reagents by inserting a needle orplastic tube into the reagent reservoir or vial, drawing up the reagentinto the tube with a motor driven syringe, moving the needle to theslide (or other receptacle) and reversing the syringe to dispense thereagent. In a process such as the foregoing, many of the reagents mustbe deposited on the slide in precisely measured small amounts (as low asthe microliter scale).

Instrumentation such as the Ventana Medical Systems ES®, NexES®,BENCHMARK® and DISCOVERY® systems are fundamentally designed tosequentially apply reagents to tissue sections mounted on one bythree-inch glass microscope slides under controlled environmentalconditions. The instrument must perform several basic functions such asreagent application, washing (to remove a previously applied reagent),jet draining (a technique to reduce the residual buffer volume on aslide subsequent to washing), Liquid Coverslip™ application (a light oilapplication used to contain reagents and prevent evaporation), and otherinstrument functions.

The process of staining tissue on a slide consists of the sequentialrepetition of the basic instrument functions described above.Essentially a reagent is applied to the tissue then incubated for aspecified time at a specific temperature. When the incubation time iscompleted the reagent is washed off the slide and the next reagent isapplied, incubated, and washed off, etc., until all of the reagents havebeen applied and the staining process is complete.

BRIEF SUMMARY OF THE DISCLOSURE

In one aspect of the present disclosure is a primary stain compositioncomprising a dye (e.g. hematoxylin or eosin), a surfactant, and aviscosity modifier. In some embodiments, the primary stain compositionis suitable for dispensing from a droplet-on-demand reagent dispensingsystem as described further herein. In some embodiments, thedroplet-on-demand reagent dispensing system is an inkjet dispensingsystem. In some embodiments, the primary stain composition furthercomprises aluminum chloride.

In another aspect of the present disclosure is a primary staincomposition comprising a dye, a surfactant, and a viscosity modifier,wherein the composition has a viscosity ranging from about 1 cp to about40 cp, and a surface tension ranging from about 25 dyne/cm to about 45dyne/cm. In some embodiments, the composition has a viscosity rangingfrom about 6 cp to about 10 cp. In some embodiments, the dye is selectedfrom the group consisting of hematoxylin, eosin acridine orange, bismarkbrown, carmine, coomassie blue, cresyl violet, crystal violet, DAPI(“2-(4-Amidinophenyl)-1H-indole-6-carboxamidine”), ethidium bromide,acid fucsine, Hoechst stains, iodine, malachite green, methyl green,methylene blue, neutral red, nile blue, nile red, osmium tetraoxide,rhodamine, and safranine. In some embodiments, the surfactant is anon-ionic surfactant. In some embodiments, the viscosity modifier is aglycol. In some embodiments, the viscosity modifier is a propyleneglycol. In some embodiments, the surfactant is present in an amountranging from between about 0.01% to about 0.5% by total weight of theprimary stain composition. In some embodiments, the viscosity modifieris present in an amount ranging from between about 35% to about 60% bytotal weight of the primary stain composition. In some embodiments, theprimary stain composition further comprises a buffer. In someembodiments, the primary stain composition further comprises a buffer.In some embodiments, the primary stain composition has a pH ranging fromabout 2 to about 5. In some embodiments, the primary stain compositionhas a pH of about 2.2. In some embodiments, the primary staincomposition further comprises aluminum chloride.

In some embodiments, the dye is hematoxylin, the surfactant is anon-ionic surfactant, the viscosity modifier is propylene glycol; andthe amount of propylene glycol ranges from about 35% to about 60% bytotal weight of the primary stain composition. In some embodiments, thedye is eosin, the surfactant is a non-ionic surfactant, the viscositymodifier is propylene glycol; and the amount of propylene glycol rangesfrom about 35% to about 60% by total weight of the primary staincomposition.

In another aspect of the present disclosure is a kit comprising a firstprimary stain composition and a second primary stain composition,wherein the first primary stain composition comprises hematoxylin, anon-ionic surfactant, and propylene glycol, wherein the propylene glycolis present in an amount ranging from about 35% to about 60% by totalweight of the first primary stain composition; and wherein the secondprimary stain composition comprises eosin, a non-ionic surfactant, andpropylene glycol, wherein the propylene glycol is present in an amountranging from about 35% to about 60% by total weight of the secondprimary stain composition.

In another aspect of the present disclosure is a large molecule reagentcomposition comprising a biological molecule selected from the groupconsist of an antibody, an antibody conjugate, an enzyme, and amultimer; a surfactant; and a viscosity modifier; wherein the antibodycomposition is suitable for dispensing from a droplet-on-demand reagentdispensing system as described further herein. In some embodiments,droplet-on-demand reagent dispensing system is an inkjet dispensingsystem. In some embodiments, the large molecule reagent compositionfurther comprises aluminum chloride.

In another aspect of the present disclosure is an antibody stainingcomposition comprising an antibody or antibody conjugate, a surfactant,and a viscosity modifier, wherein the antibody composition has aviscosity ranging from about 4 cp to about 11 cp, and a surface tensionranging from about 20 dyne/cm to about 40 dyne/cm. In some embodiments,the antibody staining composition further comprises at least one carrierprotein. In some embodiments, at least one carrier protein is selectedfrom the group consisting of bovine serum albumin and normal goat serum.In some embodiments, the surfactant is a non-ionic surfactant. In someembodiments, the surfactant is present in an amount ranging from about0.01% to about 0.5% by total weight of the antibody stainingcomposition. In some embodiments, the viscosity modifier is glycerol. Insome embodiments, the viscosity modifier is present in an amount rangingfrom about 2% to about 50% by total weight of the antibody stainingcomposition. In some embodiments, the surfactant is a non-ionicsurfactant, the viscosity modifier is glycerol or high molecular weightdextran, and wherein the composition further comprises bovine serumalbumin; and wherein an amount of glycerol or high molecular weightdextran ranges from about 2% to about 50% by total weight of theantibody staining composition. In some embodiments, the large moleculereagent composition further comprises aluminum chloride.

In another aspect of the present disclosure is a kit comprising a firstcomposition comprising an antibody staining composition and a secondcomponent comprising at least one primary stain composition. In someembodiments, the primary stain composition comprises one of hematoxylinor eosin, a non-ionic surfactant, and propylene glycol, wherein thepropylene glycol is present in an amount ranging from about 35% to about60% by total weight of the primary stain composition. In someembodiments, the antibody composition comprises a primary antibody, asurfactant, and a viscosity modifier, wherein the antibody compositionhas a viscosity ranging from about 4 cp to about 11 cp, and a surfacetension ranging from about 20 dyne/cm to about 40 dyne/cm.

In another aspect of the present disclosure is a method of staining atissue sample comprising (a) positioning a droplet-on-demand print head(e.g. an inkjet print head or other droplet dispensing means) inproximity to (e.g. near, over, or around in x,y,z, space) a portion ofthe tissue sample to receive a staining reagent, the print head influidic communication with a source of the staining reagent; (b)dispensing a predetermined amount the staining reagent from the inkjetprint head and onto the portion of the tissue sample at a predeterminedvelocity. In some embodiments, the method comprises repeating step (b)one or more times. In some embodiments, the method comprises repeatingstep (b) at least three times. In some embodiments, the method isrepeated for different portions of the tissue sample.

In some embodiments, the method further comprises measuring a stainingintensity of the dispensed staining reagent. In some embodiments, step(b) is repeated if the measured staining intensity does not meet apredetermined threshold. In some embodiments, the predeterminedthreshold is an absorbance value of between about 30 AU (arbitraryunits) and about 160 AU. In some embodiments, the predeterminedthreshold is an absorbance value of between about 25 AU and about 60 AU.In some embodiments, the predetermined threshold is an absorbance valueof between about 30 AU and about 70 AU. In some embodiments, thepredetermined threshold is an absorbance value of between about 44 AUand about 145 AU.

In some embodiments, step (b) is repeated until a cumulative amount ofthe staining reagent ranges from about 10 μL/in² to about 30 μL/in². Insome embodiments, step (b) is repeated until a cumulative amount of thestaining reagent ranges from about 12 μL/in² to about 28 μL/in². In someembodiments, step (b) is repeated until a cumulative amount of thestaining reagent ranges from about 14 μL/in² to about 28 μL/in².

In some embodiments, the dispensing method replenishes a stain depletionlayer in communication with the portion of the tissue sample. In someembodiments, the predetermined velocity is one which allows for thestaining reagent to penetrate a puddle in communication with the tissuesample and to replenish a stain depletion layer. In some embodiments,the predetermined velocity is one which allows mixing at an interfaciallayer of the tissue sample. In some embodiments, the predeterminedvelocity ranges from about 5 m/s to about 15 m/s.

In some embodiments, two staining reagents are sequentially applied tothe tissue sample. In some embodiments, the staining reagent is aprimary stain. In some embodiments, the staining reagent is acomposition comprising a dye, a surfactant, and a viscosity modifier,wherein the composition has a viscosity ranging from about 1 cp to about40 cp and a surface tension ranging from about 25 dyne/cm to about 45dyne/cm. In some embodiments, the composition is dispensed at a shearrate of between about 1×10⁵ s⁻¹ and about 1×10⁷ s⁻¹. In someembodiments, one of hematoxylin or eosin is applied first to at leastthe portion of the tissue sample, and subsequently another ofhematoxylin or eosin is applied second to at least the same portion ofthe tissue sample.

In some embodiments, the staining reagent is a large molecule stainingcomposition. In some embodiments, the large molecule stainingcomposition comprises a large molecule selected from the groupconsisting of an antibody, an antibody conjugate, a multimer, and anenzyme; a surfactant; and a viscosity modifier, wherein the compositionhas a viscosity ranging from about 4 cp to about 11 cp, and a surfacetension ranging from about 20 dyne/cm to about 40 dyne/cm. In someembodiments, the large molecule staining composition is dispensed at ashear rate of less than about 5×10⁵

In some embodiments, the method further comprises the step of optionallydepositing one or more additional reagents prior to or after eachdispensing step, wherein the one or more additional reagents areselected from the group consisting of deparaffinization agents, washes,rinses, diluents, buffers, or detection reagents. In some embodiments,the step of optionally depositing one or more reagents is performed bydispensing the one or more reagents from an inkjet print head. In someembodiments, the step of optionally depositing one or more reagents isperformed by another deposition means.

In another aspect of the present disclosure is a method of dispensingreagent onto a biological sample comprising: overlaying a protectivefluid layer onto a biological sample, the biological sample disposed ona support medium; dispensing reagent droplets of between about 1 pL toabout 50 pL such that the reagent droplets penetrate the protectivefluid layer and contact the biological sample; wherein the reagentdroplets comprise a reagent composition selected from the groupconsisting of a primary stain reagent composition and an antibodyreagent composition. In some embodiments, the reagent droplets aredispensed at a velocity of between about 5 m/s to about 15 m/s. In someembodiments, the protective fluid layer is an aqueous puddle. In someembodiments, the protective fluid layer is an immiscible oil. In someembodiments, a density of the reagent droplets is greater than a densityof the immiscible oil. In some embodiments, a kinetic energy of thereagent droplets is greater than a surface tension of the immiscibleoil. In some embodiments, a kinetic energy of the reagent droplets isgreater than a surface tension of the protective fluid layer. In someembodiments, the kinetic energy is greater than 9.52×10⁻¹⁰ Joules.

In some embodiments, the primary stain reagent composition comprises adye, a surfactant, and a viscosity modifier, wherein the composition hasa viscosity ranging from about 1 cp to about 40 cp and a surface tensionranging from about 25 dyne/cm to about 45 dyne/cm. In some embodiments,the primary stain reagent composition is dispensed at a shear rate ofbetween about 1×10⁵ s⁻¹ and about 1×10⁷ s⁻¹. In some embodiments, theantibody reagent composition comprising a primary antibody, asurfactant, and a viscosity modifier, wherein the composition has aviscosity ranging from about 4 cp to about 7 cp, and a surface tensionranging from about 20 dyne/cm to about 40 dyne/cm. In some embodiments,the antibody composition is dispensed at a shear rate of less than about5×10⁵ s⁻¹.

In another aspect of the present disclosure is a method of dispensingreagent onto a biological sample comprising: overlaying a protectivefluid layer onto a biological sample, the biological sample disposed ona support medium; dispensing a pH modifier to the biological sample; anddispensing reagent droplets at a velocity of between about 5 m/s toabout 15 m/s; wherein the reagent droplets comprise a reagentcomposition selected from the group consisting of a primary stainreagent composition and an antibody reagent composition. In someembodiments, an amount of reagent droplets dispensed ranges from about10 μL/in² to about 30 μL/in². In some embodiments, the pH modifier has apH ranging from about 3 to about 5. In some embodiments, the protectivefluid layer is an immiscible oil and wherein a kinetic energy of thereagent droplets is greater than a surface tension of the immiscibleoil. In some embodiments, the primary stain reagent compositioncomprises a dye, a surfactant, and a viscosity modifier, wherein thecomposition has a viscosity ranging from about 1 cp to about 40 cp and asurface tension ranging from about 25 dyne/cm to about 45 dyne/cm. Insome embodiments, primary stain reagent composition is dispensed at ashear rate of between about 1×10⁵ s⁻¹ and about 1×10⁷ s⁻¹. In someembodiments, an antibody reagent composition comprising a primaryantibody, a surfactant, and a viscosity modifier, wherein thecomposition has a viscosity ranging from about 4 cp to about 7 cp, and asurface tension ranging from about 20 dyne/cm to about 40 dyne/cm. Insome embodiments, the antibody composition is dispensed at a shear rateof less than about 5×10⁵ s⁻¹.

In another aspect of the present disclosure is a method of dispensingreagent onto a biological sample comprising: overlaying a protectivefluid layer onto a biological sample, the biological sample disposed ona support medium; dispensing reagent droplets with a kinetic energysufficient to penetrate the protective fluid layer for the reagentdroplets to reach the biological sample and such that a spatial densityof reagent droplets deposited on the biological sample ranges from about50 dpi to about 1200 dpi, wherein the reagent droplets comprise areagent composition selected from the group consisting of a primarystain reagent composition and a large molecule staining composition. Insome embodiments, the protective fluid layer is an immiscible oil andwherein a density of the reagent droplets is greater than a density ofthe immiscible oil. In some embodiments, the large molecule stainingcomposition comprises a large molecule selected from the groupconsisting of an antibody, an antibody conjugate, a multimer, and anenzyme; a surfactant; and a viscosity modifier, wherein the compositionhas a viscosity ranging from about 4 cp to about 7 cp, and a surfacetension ranging from about 20 dyne/cm to about 40 dyne/cm. In someembodiments, the large molecule staining composition is dispensed at ashear rate of less than about 5×10⁵ s⁻¹. In some embodiments, theprimary stain reagent composition comprises a dye, a surfactant, and aviscosity modifier, wherein the composition has a viscosity ranging fromabout 1 cp to about 40 cp and a surface tension ranging from about 25dyne/cm to about 45 dyne/cm. In some embodiments, the primary stainreagent composition is dispensed at a shear rate of between about 1×10⁵s⁻¹ and about 1×10⁷ s⁻¹.

In another aspect of the present disclosure is a method of dispensingone or more reagents onto a biological sample, the method comprisingoverlaying a protective fluid layer onto a biological sample, thebiological sample disposed on a support medium (e.g. a microscopeslide); dispensing reagent droplets via a droplet-on-demand system suchthat the reagent droplets penetrate the protective fluid layer andcontact the biological sample; wherein the reagent is selected from thegroup consisting of a primary stain reagent composition or a largemolecule reagent reagent composition. In some embodiments, the primarystain composition comprises a dye, a surfactant, and a viscositymodifier, wherein the composition has a viscosity ranging from about 1cp to about 40 cp and a surface tension ranging from about 25 dyne/cm toabout 45 dyne/cm. In some embodiments, the dye is hematoxylin, thesurfactant is a non-ionic surfactant, the viscosity modifier ispropylene glycol; and wherein an amount of propylene glycol ranges fromabout 35% to about 60% by total weight of the primary stain composition.In some embodiments, the dye is eosin, the surfactant is a non-ionicsurfactant, the viscosity modifier is propylene glycol; and wherein anamount of propylene glycol ranges from about 35% to about 60% by totalweight of the primary stain composition. In some embodiments, the largemolecule reagent staining composition comprises a primary antibody, asurfactant, and a viscosity modifier, wherein the composition has aviscosity ranging from about 4 cp to about 7 cp, and a surface tensionranging from about 20 dyne/cm to about 40 dyne/cm. In some embodiments,the protective fluid layer is an aqueous puddle. In some embodiments,the reagent droplets are provided with a velocity sufficient topenetrate and replenish a depletion layer around the biological sample.In some embodiments, the reagent droplets are dispensed at a velocity ofbetween about 5 m/s to about 15 m/s. In some embodiments, the protectivefluid layer is an immiscible fluid, e.g. an oil. In some embodiments, adensity of the reagent droplets is greater than a density of theimmiscible oil. In some embodiments, a kinetic energy of the reagentdroplets is greater than a surface tension of the immiscible oil. Insome embodiments, a Weber number of the reagent droplets is less thanabout 18. In some embodiments, the primary stain reagent solution isdispensed at a shear rate of between about 1×10⁵ s⁻¹ and about 1×10⁷ s⁻¹and the antibody reagent solution is dispensed at a shear rate of lessthan about 5×10⁵ s⁻¹.

In another aspect of the present disclosure is a means for dispensing aprimary staining reagent composition or a large molecule reagentstaining composition to a tissue sample, wherein a volume of the primarystaining reagent composition or the large molecule reagent stainingcomposition dispensed ranges from about 10 μL/in² to about 30 μL/in². Insome embodiments, the dispensing means is an inkjet print head. In someembodiments, the dispensing means is a droplet-on-demand system,comprising a target imaging system, a relative motion system, a printhead, a fluid reservoir, and a pressure control means. In someembodiments, the dispensing means further comprises a print headcleaning system. In some embodiments, the dispensing means is asillustrated in FIG. 1A. In some embodiments, the primary stainingreagent composition and the large molecule reagent staining compositionare formulated to have a viscosity suitable for dispensing with thedispensing means.

In another aspect of the present disclosure is an automated slidestaining apparatus comprising (a) a dispenser for dispensing reagentdroplets having a volume ranging from about 1 pL to about 50 pL; (b) aslide support adapted to hold a microscope slide; (c) at least onereagent reservoir comprising a primary stain reagent composition or alarge molecule stain composition, the reservoir in fluid communicationwith the inkjet printing head; and (d) a control module containing aprocessor and memory, wherein the control module is programmed to directthe inkjet printing head to dispense the composition onto a microscopeslide held by the slide support.

In another aspect of the present disclosure is a computer implementedmethod comprising the steps of (i) imaging a first portion of a slide,the slide containing a tissue sample; (ii) selecting a second portion ofthe slide for application of a staining reagent, wherein the secondportion is a subset of the first portion; (iii) depositing a stainingreagent to the second portion via an inkjet deposition system over aplurality of passes. In some embodiments, between about 360 nL/in² toabout 14.4 μL/in² of staining reagent is deposited to the second portionper pass of the deposition system.

In another aspect of the present disclosure is a computer system forstaining a tissue sample comprising one or more processors and at leastone memory, the at least one memory storing non-transitorycomputer-readable instructions for execution by the one or moreprocessors to cause a staining apparatus, having a droplet-on-demanddispensing mechanism, in communication with the computer system todispense a predetermined quantity of a primary staining reagentcomposition or a large molecule reagent composition onto at least aportion of a biological sample.

It would be advantageous to apply or dispense reagents with moreprecision (e.g. dosing precision, temporal precision, in situ mixing) ascompared with conventional staining methods. It would also beadvantageous to dispense reagents with less reagent volume (and henceless waste), and/or to drive staining kinetics at higher rates, again ascompared with conventional staining methods. Applicants have found thatdispensing of reagent solutions via a droplet-on-demand technology (e.g.inkjet technology or piezoelectric technology) enables consistentresults, and is suitable for incorporation within automated stainingprocesses. Applicants have also discovered that a staining intensity maybe optimized (e.g. “dialed-in”) by dispensing more or less reagent massonto the tissue via a droplet-on-demand system. Indeed, Applicants havefound that reagent mass may be varied by one of several methodsincluding (i) applying reagent by multiple passes of a dispensingmechanism; (ii) varying the dots per inch (dpi) of reagent dispensing;(iii) varying the droplet volume; and/or (iv) varying the reagentconcentration, as disclosed herein. Applicants have unexpectedlydiscovered that the staining reaction kinetics appear to be faster thanwith prior art puddle technology, as discussed further herein.Applicants have also found that dispensing reagent solutions via adroplet-on-demand deposition process allows for a significant reductionin reagent usage, again while providing the same staining intensities ascompared with conventional staining methods. These and othercomparatively superior results are described further herein.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided to the Office upon request and thepayment of the necessary fee.

FIG. 1A sets forth a droplet-on-demand system according to embodimentsof the present disclosure;

FIG. 1B sets forth a system, which may comprise or be tied to adroplet-on-demand system according to embodiments of the presentdisclosure;

FIG. 2 sets forth a reagent dispensing process according to oneembodiment of the present disclosure;

FIG. 3 sets forth a reagent dispensing process according to anotherembodiment of the present disclosure;

FIGS. 4A through 4E illustrate staining of a tissue sample withhematoxylin, whereby the tissue was stained with a reagent dispensingprocess according to one embodiment of the present disclosure, andfurther compares droplet-on-demand staining with conventional stainingtechniques;

FIGS. 5A through 5C illustrate staining of a tissue sample with eosin,whereby the tissue was stained with a reagent dispensing processaccording to one embodiment of the present disclosure, and furthercompares droplet-on-demand staining with conventional stainingtechniques;

FIGS. 6A and 6B illustrate staining of a tissue sample with bothhematoxylin and eosin using a reagent dispensing process according toone embodiment of the present disclosure;

FIG. 7 sets forth a flowchart illustrating one process for staining atissue sample with a primary stain or an antibody in an IHC process;

FIG. 8A sets forth a reagent dispensing process according to oneembodiment of the present disclosure;

FIG. 8B illustrates a tissue sample staining using a reagent dispensingprocess according to one embodiment of the present disclosure;

FIGS. 9A, 9B, and 9C illustrate staining achieved through deposition ofprimary antibodies using a reagent dispensing process according to oneembodiment of the present disclosure;

FIGS. 10A through 10C illustrate additional examples of the depositionof primary antibodies using a reagent dispensing process according toone embodiment of the present disclosure;

FIG. 11 illustrates staining of different tissue regions with differentreagents;

FIG. 12A compares the ratio of staining intensity in cell nuclei to thestain intensity of cytoplasmic and extracellular regions of tissue; and

FIG. 12B illustrates the effects of staining at different pHs.

DETAILED DESCRIPTION

In general, the present disclosure is directed to the delivery ordispensing of one or more reagents to a biological sample utilizingdroplet-on-demand technology. Once dispensed, the reagent is distributedto the cells, cell membranes, nuclei and/or tissue or structurescontained within the biological sample. The presently disclosed methodis uniquely characterized by the ability to deposit reagents forstaining reactions with (i) spatial selectivity both within a sample andon a slide; (ii) the ability to print films onto the sample with reagentfilm thicknesses down to the size scale of a tissue section(approximately 4 μm in height) which is smaller than the thickness ofany diffusion depletion layers that exist in staining puddles; and (iii)the ability to stain regions of interest on the sample down to the sizescale of single droplets, as defined by the particular dropletgeneration technology utilized. For example, in some embodiments, theminimum staining region is an area of tissue approximately 25-60 μm indiameter. As will be described in further detail herein, the reagentdispensed include a primary stain or an antibody useful inhistochemistry, such that targets within the biological sample may bestained, detected and analyzed.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless the context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise.

The terms “comprising,” “including,” “having,” and the like are usedinterchangeably and have the same meaning. Similarly, “comprises,”“includes,” “has,” and the like are used interchangeably and have thesame meaning. Specifically, each of the terms is defined consistent withthe common United States patent law definition of “comprising” and istherefore interpreted to be an open term meaning “at least thefollowing,” and is also interpreted not to exclude additional features,limitations, aspects, etc. Thus, for example, “a device havingcomponents a, b, and c” means that the device includes at leastcomponents a, b and c. Similarly, the phrase: “a method involving stepsa, b, and c” means that the method includes at least steps a, b, and c.Moreover, while the steps and processes may be outlined herein in aparticular order, the skilled artisan will recognize that the orderingsteps and processes may vary.

As used herein, the term “antibody,” refers to immunoglobulins orimmunoglobulin-like molecules, including by way of example and withoutlimitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, andsimilar molecules produced during an immune response in any vertebrate,(e.g., in mammals such as humans, goats, rabbits and mice) and antibodyfragments (such as F(ab′)2 fragments, Fab′ fragments, Fab′-SH fragmentsand Fab fragments as are known in the art, recombinant antibodyfragments (such as sFv fragments, dsFv fragments, bispecific sFvfragments, bispecific dsFv fragments, F(ab)′2 fragments, single chain Fvproteins (“scFv”), disulfide stabilized Fv proteins (“dsFv”), diabodies,and triabodies (as are known in the art), and camelid antibodies) thatspecifically bind to a molecule of interest (or a group of highlysimilar molecules of interest) to the substantial exclusion of bindingto other molecules. Antibody further refers to a polypeptide ligandcomprising at least a light chain or heavy chain immunoglobulin variableregion which specifically recognizes and binds an epitope of an antigen.Antibodies may be composed of a heavy and a light chain, each of whichhas a variable region, termed the variable heavy (VH) region and thevariable light (VL) region. Together, the VH region and the VL regionare responsible for binding the antigen recognized by the antibody. Theterm antibody also includes intact immunoglobulins and the variants andportions of them well known in the art.

As used herein, the term “antigen” refers to a compound, composition, orsubstance that may be specifically bound by the products of specifichumoral or cellular immunity, such as an antibody molecule or T-cellreceptor. Antigens can be any type of molecule including, for example,haptens, simple intermediary metabolites, sugars (e.g.,oligosaccharides), lipids, and hormones as well as macromolecules suchas complex carbohydrates (e.g., polysaccharides), phospholipids, nucleicacids and proteins.

A “biological sample” or “tissue sample” can be any solid or fluidsample obtained from, excreted by or secreted by any living organism,including without limitation, single celled organisms, such as bacteria,yeast, protozoans, and amoebas among others, multicellular organisms(such as plants or animals, including samples from a healthy orapparently healthy human subject or a human patient affected by acondition or disease to be diagnosed or investigated, such as cancer)which are suitable for histochemical or cytochemical analysis, such assamples that preserve the morphological characteristics of the cellsand/or tissues to be analyzed. For example, a biological sample can be abiological fluid obtained from, for example, blood, plasma, serum,urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreoushumor, or any bodily secretion, a transudate, an exudate (for example,fluid obtained from an abscess or any other site of infection orinflammation), or fluid obtained from a joint (for example, a normaljoint or a joint affected by disease). A biological sample can also be asample obtained from any organ or tissue (including a biopsy or autopsyspecimen, such as a tumor biopsy) or can include a cell (whether aprimary cell or cultured cell) or medium conditioned by any cell, tissueor organ. In some examples, a biological sample is a nuclear extract. Incertain examples, a sample is a quality control sample. In otherexamples, a sample is a test sample. For example, a test sample is acell, a tissue or cell pellet section prepared from a biological sampleobtained from a subject. In an example, the subject is one that is atrisk or has acquired. Samples can be prepared using any method known inthe art by of one of ordinary skill. The samples can be obtained from asubject for routine screening or from a subject that is suspected ofhaving a disorder, such as a genetic abnormality, infection, or aneoplasia. The described embodiments of the disclosed method can also beapplied to samples that do not have genetic abnormalities, diseases,disorders, etc., referred to as “normal” samples. Samples can includemultiple targets that can be specifically bound by one or more detectionprobes.

As used herein, the phrase “dip and dunk” refers to a stainingtechnology whereby the sample and microscope slide are submerged into apath of staining reagent for each assay step.

As used herein, the terms “drop-on-demand,” “droplet-on-demand”, or“droplet-based” (and other like terms or phrases) refer to a stainingtechnology that deposits discrete droplets of reagent onto the targetsample, as opposed to “flooding” the slide or sample thereon withreagent. In some embodiments, the droplet-on-demand technology utilizesinkjet technology or piezoelectric technology. In some embodimentsdisclosed herein, the droplet dispensing technology is facilitated usingan inkjet print head or like technology.

As used herein, the term “humectant” refers to a hygroscopic substanceused to keep a substance, e.g. a tissue sample, moist; it is theopposite of a desiccant. It is often a molecule with several hydrophilicgroups, most often hydroxyl groups; however, amines and carboxyl groups,sometimes esterified, can be encountered as well (its affinity to formhydrogen bonds with molecules of water is the crucial trait). It isbelieved that a humectant attracts and retains the moisture in the airnearby via absorption, drawing the water vapor into and/or beneath theorganism/object's surface. By contrast, desiccants also attract ambientmoisture, but adsorb—not absorb—it, by condensing the water vapor ontothe surface, as a layer of film. In the context of inkjet deposition orlike technologies, a humectant may be important for maintaining a viablenozzle. In some embodiments, it is important for keeping the tissuesample or biological sample hydrated during thin film processing.

The term “inkjet” in this disclosure refers to the family ofdrop-on-demand technologies where a piezoelectric (or thermal) elementis used to actuate a droplet from a dispense manifold. This may includedirect and non-contact methods common to the commercial printingindustry or those used outside of the commercial printing industry.

As used herein, the term “immunohistochemistry” refers to a method ofdetermining the presence or distribution of an antigen in a sample bydetecting interaction of the antigen with a specific binding agent, suchas an antibody. A sample is contacted with an antibody under conditionspermitting antibody-antigen binding. Antibody-antigen binding can bedetected by means of a detectable label conjugated to the antibody(direct detection) or by means of a detectable label conjugated to asecondary antibody, which binds specifically to the primary antibody(indirect detection).

As used herein, the term “microfluidic” refers to a staining technologyrequiring an opposable surface to the glass microscope slide and a meansfor a flow-based introduction and evacuation of staining reagents intothe gap. Further, a desired gap height should be created such that theflow of reagent across the surface of the slide is laminar and the totalvolume inside of the gap is minimized.

As used herein, the term “primary antibody” refers to an antibody whichbinds specifically to a target protein antigen in a tissue sample. Aprimary antibody is generally the first antibody used in animmunohistochemical procedure. Primary antibodies also include thoseantibodies conjugated to another molecule (e.g. a label, hapten, etc.).Primary antibodies may serve as “detection probes” for detecting atarget within a tissue sample.

As used herein, the term “primary stain” is a dye or like molecule thatenhances contrast in a tissue sample. In some embodiments, the primarystain is one which directly “labels” a biological structure within or ona cell, without the employment of a specific binding agent, such as anantibody. Some examples of primary stains include hematoxylin and eosin.Other examples of primary stains include acridine orange, bismark brown,carmine, coomassie blue, cresyl violet, crystal violet, DAPI(“2-(4-Amidinophenyl)-1H-indole-6-carboxamidine”), Ethidium bromide,acid fucsine, Hoechst stains (Hoechst 33342 and Hoechst 33258, which area bis-benzimidazole derivatives), iodine, malachite green, methyl green,methylene blue, neutral red, nile blue, nile red, osmium tetraoxide,rhodamine, and safranin. Other examples of primary stains include thosestain used to stain bacteria (Gram-positive or Gram-negative stains),stains used to identify endospores (endospore staining), stains used tohelp identify species of Mycobacterium tuberculosis (Ziehl-Neelsenstain), Papanicolaou staining kits (which use a combination ofhaematoxylin, Orange G, eosin Y, Light Green SF yellowish, and sometimesBismarck Brown Y), Periodic acid-Schiff stains (“PAS stains”), silverstains, etc. Yet other non-limiting primary stains include (i)histologic stains to selectively demonstrate Mycobacterium and otheracid fast organisms or components (e.g. the AFB III Staining Kit,available from Ventana Medical Systems, Tucson, Ariz.); (ii) histologicstains to differentiate acid mucin from neutral polysaccharides (e.g.the Alcian Blue for PAS, also available from Ventana); (iii) histologicstain sto demonstrate weakly acidic mucopolysaccharide (e.g. Alcian BlueStaining Kit, also available from Ventana); (iv) histologic stains forHelicobacter pylori (e.g. Alcian Yellow Staining Kit, also availablefrom Ventana); (v) histologic stains to selectively demonstrate amyloid(e.g. Congro Red Staining Kit, also available from Ventana); (vi)histologic stains to differentiate acid mucin from neutralpolysaccharides (e.g. Diastase Kit, also available from Ventana); (vii)histologic stains to demonstrate elastic fibers in tissue sections (e.g.Elastic Staining Kit, also available from Ventana); (viii) histologicstains to differentiate leukocytes in bone marrow and otherhematopoietic tissue (lymph nodes) (e.g. Giemsa Staining Kit, alsoavailable from Ventana); (ix) histologic stains to demonstratepolysaccharides in the cell walls of fungi and other opportunisticorganisms, including, but not limited to, stains able to distinguishpathogenic fungi such as Aspergillus and Blastomyces1 and otheropportunistic organisms such as Pneumocystis carinii (e.g. GMS IIStaining Kit, also available from Ventana); (x) histologic stains todemonstrate gram-negative and gram-positive bacteria (e.g. Gram StainingKit, also available from Ventana); (xi) histologic stains used to studyconnective tissue, muscle and collagen fibers (e.g. Green for Trichrome,also available from Ventana); (xii) histologic stains to detect ironpigment in bone marrow, tissue with hemochromatosis, and hemosiderosis(e.g. Iron Staining Kit, also available from Ventana); (xiii) histologicstains to demonstrate capillary basement membrane (e.g. Jones H&EStaining Kit or Jones Light Green Statining kit, both also availablefrom Ventana); (xiv) histologic stains for detection of fungus (e.g.Light Green for PAS, also available from Ventana); (xv) histologicstains to detect acid mucopolysaccharides (mucin) (e.g. MuciarmineStaining Kit, also available from Ventana); (xvi) histologic stains usedto demonstrate the presence of glycogen, including stains that mayassist in the identification of positive reticular fibers, basementmembrane, fungus, and neutral mucopolysaccharides, or those stains thatmay aid in distinguishing a PAS positive secreting adenocarcinoma froman undifferentiated PAS negative squamous cell carcinoma (e.g. PASStaining Kit, also available from Ventana); (xvii) histologic stains todemonstrate reticular fiber (e.g. Reticulum II Staining Kit, alsoavailable from Ventana); (xviii) histologic stains used to studyspecific argyrophilic microorganisms (e.g. Steiner II Staining Kit, alsoavailable from Ventana); (xix) histologic silver stains to aide in theidentification of the causative organisms of diseases such as somegastric ulcers (H. pylori), Lyme disease, Legionnaire's disease, catscratch fever, etc. (e.g. Steiner Staining Kit, also available fromVentana); (xx) histologic stains to study connective tissue, muscle andcollagen fibers (e.g. Trichrome II Blue Staining Kit, also availablefrom Ventana); (xxi) histologic stains to study connective tissue,muscle and collagen fibers (e.g. Trichrome Staining Kit, Trichrome IIIBlue Staining Kit, or Trichrome III Green Staining Kit, each alsoavailable from Ventana). The skilled artisan will also recognize thatthere exist other primary stains, or for that matter dyes, that may beused in conjunction with the kits, methods, and compositions (e.g.primary stain compositions, reagent compositions) of the presentdisclosure.

As used herein, the term “puddle” refers to a single-slide stainingtechnology whereby the entire sample area surface of the microscopeslide is covered in a volume of reagent for each assay step.

As used herein, the term “reagent” may refer to any fluid deposited ontoa tissue section or cytology sample, that is used in the context of amorphological (e.g. hematoxylin and eosin), immunohistochemical, orspecial stain. This includes, but is not limited to, oils, organics, andbridging reagents for removing wax (i.e. deparaffinization); washes,rinses, diluents, or buffers used to set reaction conditions, dilutereagents to an appropriate concentration, quench reactions, or wash awayexcess reactants; small molecule dyes used for morphological stainingand special stains; antibodies, antibody conjugates, enzymes, multimers,amplifiers, chromogenic substrates, fluorescent detection chemistries,chemiluminescent substrates, and enzyme-reaction co-factors, used in IHCor ICC staining.

As used herein, “surfactants” are classified as anionic, cationic, ornonionic, depending on their mode of chemical action. In general,surfactants reduce interfacial tension between two liquids. A surfactantmolecule typically has a polar or ionic “head” and a nonpolarhydrocarbon “tail.” Upon dissolution in water, the surfactant moleculesaggregate and form micelles, in which the nonpolar tails are orientedinward and the polar or ionic heads are oriented outward toward theaqueous environment. The nonpolar tails create a nonpolar “pocket”within the micelle. Nonpolar compounds in the solution are sequesteredin the pockets formed by the surfactant molecules, thus allowing thenonpolar compounds to remain mixed within the aqueous solution. In someembodiments, the surfactant may be used to produce uniform spreading ofreagents across a tissue section as well as decrease backgroundstaining.

As used herein, a “target” may be a particular tissue in a biologicalsample or a particular molecule or marker in a biological sample.Examples of the target include antigens (including haptens), antibodies,and enzymes. Further examples of targets include, generally, proteins,peptides, nucleic acids, sugars, and lipids. The reagents for use in thepresent disclosure may be those that are capable of converting thetarget materials present in the biological sample into detectable formsso that the localization of the targets can be detected (such asvisually).

Droplet-on-Demand Dispensing System and Methods of Dispensing Reagents

In one aspect of the present disclosure is a device or system for thedeposition of one or more reagents onto biological samples comprising areagent deposition system utilizing droplet-on-demand technology, e.g.an inkjet dispensing system. According to the present disclosure, thereagent, or composition comprising the reagent, is delivered onto thebiological sample, or a region or portion thereof, in the form ofdroplets to effect spotting or staining of the sample with the reagentsolution. Droplet-on-demand technology, including piezoelectric orinkjet dispensing technology, is described further in, for example, U.S.Pat. No. 4,877,745 and in PCT Publication No. WO98/47006, thedisclosures of which are hereby incorporated by reference herein intheir entireties.

The elements of a droplet-on-demand staining system 900 according tosome embodiments of the present disclosure are set forth in FIG. 1A.FIG. 1A illustrates the print head 905 and its relationship with thetarget sample 908 (the target sample may be a biological samples ortissue sample mounted on a standard microscope slide). To create a“staining job,” the target sample 908, in some embodiments, may beanalyzed by a target imaging system 901 to determine the spatialposition of the target sample. This information is then subsequently fedinto the central processing unit 902, which interprets the imaginginformation and the assay, and then subsequently sends instructionsincluding, for example, timing and coordination information, to theprint head 905, a pressure control system 903, and a relative motionsystem 904. The relative motion system 904 is designed to position theprint head 905 and/or target sample 908 into alignment to initiatedispensing of staining droplets (e.g. reagent staining droplets) ontothe sample. In some embodiments, fluid or reagent is fed from thereagent cartridge 906 to the print head 905 and onto the target sample908. The relative motion system 904 is coordinated with the dispensetiming from the print head 905 via a central processing unit 902 toproduce the desired stain image, as defined by the image provided in 901and interpreted by 902. Periodically, cleaning of a print head isrequired, per the print head cleaning station 907. Element 907 interactsdirectly with the print head to actively clean and force fluid throughthe print nozzles, priming the nozzles for staining. In someembodiments, a small positive pressure, e.g. about 10 psi or less, maybe applied to the cartridge in order to actively clear fluid from thenozzles.

The skilled artisan will appreciate that the droplet-on-demand stainingsystem 900 may be communicatively coupled to additional components, e.g.analyzers, scanners, computer systems, etc. (see FIG. 1B). In general,droplet-on-demand staining system 900 can include, without limitation, atarget imaging system 901 having one or more image capture devices.Image capture devices can include, without limitation, a camera (e.g.,an analog camera, a digital camera, etc.), optics (e.g., one or morelenses, sensor focus lens groups, microscope objectives, etc.), imagingsensors (e.g., a charge-coupled device (CCD), a complimentarymetal-oxide semiconductor (CMOS) image sensor, or the like),photographic film, or the like. In digital embodiments, the imagecapture device can include a plurality of lenses that cooperate to proveon-the-fly focusing. A CCD sensor can capture a digital image of thespecimen. One method of producing a digital image includes determining ascan area comprising a region of the microscope slide that includes atleast a portion of the specimen. The scan area may be divided into aplurality of “snapshots.” An image can be produced by combining theindividual “snapshots.” In some embodiments, the target imaging system901 produces a high-resolution image of the entire specimen, one examplefor such an apparatus being the VENTANA iScan HT slide scanner fromVentana Medical Systems, Inc. (Tucson, Ariz.), and may becommunicatively coupled to the system 900.

With reference to FIG. 1B, the computer device 14 may include a centralprocessing unit (which may be CPU 902 or may be a separate CPU), andcomputer device 14 may include a desktop computer, a laptop computer, atablet, or the like and can include digital electronic circuitry,firmware, hardware, memory, a computer storage medium, a computerprogram, a processor (including a programmed processor), or the like.The illustrated computing system 14 of FIG. 1B is a desktop computerwith a screen 16 and a tower 18. The tower 18 can store digital imagesin binary form from the target imaging system 901. In some embodiments,a network 20 or a direct connection interconnects the droplet-on-demandstaining system 900 and the computer system 14. The computer systemsinclude one or more processors that are programmed with a series ofcomputer-executable instructions, the instructions being stored in amemory.

The skilled artisan will appreciate that the droplet-on-demandcomponents may be part of a larger system comprising additionalcomponents useful in preparing, processing, and/or analyzing biologicalsamples. For example, the droplet-on-demand system 900 of the presentdisclosure may be tied to a specimen processing apparatus (eitherupstream or downstream from system 900) that can perform one or morepreparation processes on the tissue specimen. The preparation processcan include, without limitation, deparaffinizing a specimen,conditioning a specimen (e.g., cell conditioning), staining a specimen,performing antigen retrieval, etc. The skilled artisan will alsoappreciate that even though the droplet-on-demand dispensing system ofthe preset disclosure provides a means for staining a sample (e.g.primary stains or IHC stains), the system may be coupled with otherstaining systems, such as those for performing immunohistochemistrystaining (including labeling) and/or performing in situ hybridization(e.g., SISH, FISH, etc.) staining (including labeling), as well as otherprocesses for preparing specimens for microscopy, microanalyses, massspectrometric methods, or other analytical methods.

FIG. 2 sets forth a process utilizing droplet-on-demand technology aspart of an automated staining system. While FIG. 2 illustrates a processmap in the context of inkjet dispensing technology, the skilled artisanwill recognize that the system may utilize other droplet-on-demandtechnologies as known to those of ordinary skill in the art. Withreference to FIG. 2, the biological sample is first introduced 802. Insome embodiments, the biological sample may include a deparaffinizedtissue section, a frozen tissue section, or a cell sample. In someembodiments, the droplet-on-demand dispensing system may includedispensing reagents for the deparaffinization of tissue sections (ordeparaffinization may occur alternatively in an upstream process). Thenecessary assay information 801 for the incoming sample is convertedinto process steps and/or parameters and then partitioned into “inkjetstaining blocks” 803 that may be uniquely defined by hardwareconfigurations or defined by software instructions for the hardwaredefining different process blocks. The process blocks 803 may bespatially separated within a system or may exist as a monolithicarchitecture capable of reconfiguring for each of these blocks. Thecomplete assay is defined as the sequential execution of 803 processblocks with the outcome of a transformed sample ready for furtherdownstream processing 804.

FIG. 2 also provides an expanded view of one embodiment of an inkjetstaining process block 803. In some embodiments, the incoming sample isdefined as in 802 but with optional additional inkjet or droplet-basedprocessing operations performed on the sample prior to introduction tothe inkjet staining process block. Block 806 defines the front-endprocessing step where the inputs of inkjet assay information 801 and thesample 805 are synthesized and partitioned into parameter settings thatdefine the inkjet staining operation to be performed in that processblock. In some embodiments, parameters 807 to be set at this step of theprocess include (i) the particular staining reagent to be dispensed;(ii) the number of print passes to be performed over the sample; (iii)the staining area in the context of a print image file which may print(a) an entire microscope slide, (b) only the regions on the slide havingtissue samples, or (c) some combination of sample area and glassregions, multiple tissue samples, or multiple regions within a singlesample; (iv) the print DPI, defining the density of droplets to dispenseonto the target; (v) the temperature conditions of the process block;and (vi) the incubation times and routine required for the processblock. Once the process block is defined, the sample is introduced intothe block for execution of the inkjet staining process, as defined at808, 809, and 810. These three steps generally represent the activeportion of dispensing and/or the reaction of the reagents with thesample (see also FIGS. 7 and 8A). Finally, at 811 any remainingunreacted printed reagent is washed away and the excess fluid on theslide is removed resulting in a transformed sample ready for furtherdownstream processing at 804.

FIG. 3 sets forth another embodiment of an inkjet staining system, whichsets forth a staining process utilizing droplet-on-demand technology asdeveloped on a custom inkjet strainer which utilizes a DMC-11610 printhead. As will be illustrated further herein, FIG. 4A provides stainedtissue samples which were prepared according to the methods outlinedherein and as exemplified in FIG. 3. Referring again to FIG. 3, at step1401, the sample is introduced to the droplet-on-demand staining system(900) and may include a deparaffinized tissue section, a frozen tissuesection, or a cell sample. At 1401, an image of the sample is captured(e.g. use the target imaging system 901) including information about theposition of the sample with respect to the print heads. This image is,in some embodiments, converted into a stack of pixel maps, with eachpixel representing one or more droplets to be ejected from the printhead onto respective position on the sample. The stack of pixel mapsrepresents the instructions for each of the print heads to be used inthe assay, with one or more layers of the stack partitioned to each ofthe print heads. At step 1411, instructions are created and/or providedsuch that the print head may adjust the print dots-per-inch (dpi) (1412)by adjusting the sabre angle at which it prints (see US PatentPublication No. 2009/0314170 for further information regarding sabreangles, the disclosure of which is hereby incorporated by referenceherein). In some embodiments, the sabre angle sets the spacing betweenthe nozzles on the DMC-11610 print head. At the same time, the pixelmaps are also translated into a series of movements sent to the sampletransportation system. The timing of droplet deposition and the relativemovement of the sample with respect to the print head during the printoperation 1404 are coordinated by a computer system, as illustrated inFIGS. 1A and 1B.

In some embodiments, excess fluid is removed (1402) from the incomingsample. It is believed that this is an integral step leading up to thedeposition of stain with droplet deposition technologies since excessfluid may dilute the low volume of stain deposited at 1404 or lead to aninhomogeneous stain deposition. To prepare the sample for staining 1403,a deposition of non-staining fluid is used to adjust the pH and bufferconditions within the tissue sample. In this specific embodiment, thismay be performed using a bulk dispense; a print head loaded with thestaining preparation fluid; or a combination of two print heads loadedwith different buffers that ratiometrically dispense onto the tissue inorder to “dial-in” the pH conditions matched to the subsequent stainingstep(s). As an example of a ratiometric buffer system, Table 2 describesformic acid and acetic acid buffer systems that can be titrated over arange appropriate for preparing the tissue for optimal staining withhematoxylin. Steps 1404, 1405, 1406, and 1407 set forth the process forexecuting a printed stain, including printing, incubation, a feedbackloop for depositing multiple printed layers of the same stain such as to“dial-in” the intensity and specificity of the stain, as noted in FIGS.1 and 2. After a printed staining step is completed, the sample isrinsed 1407 to remove any unreacted printed reagent and either loopedback through the process 1408 to print the next staining step (e.g. aprinted eosin step subsequent to a printed hematoxylin step) or releasedfor downstream processing through an air knife (for example) step 1402to remove excess fluid after which a transformed sample is produced1409.

The skilled artisan will appreciate that the droplet-on-demanddispensing system may be “tuned” so as to provide different processingparameters depending on the type of reagent dispensed, the type ofbiological sample, or how the biological sample is prepared. As notedpreviously herein, some parameters that may be tuned include, but arenot limited to, droplet volumes, and droplet velocities. In someembodiments, the droplet-on-demand dispensing system is able to dispensebetween about 1 pL to about 10 nL of reagent per droplet of thedeposition system. In other embodiments, the droplet-on-demanddispensing system is able to dispense between about 1 pL to about 1 nLof reagent per droplet of the deposition system. In yet otherembodiments, the droplet-on-demand dispensing system is able to dispensebetween about 1 pL to about 500 pL of reagent per droplet of thedeposition system. In further embodiments, the droplet-on-demanddispensing system is able to dispense between about 1 pL to about 250 pLof reagent per droplet of the deposition system. In yet furtherembodiments, the droplet-on-demand dispensing system is able to dispensebetween about 1 pL to about 100 pL of reagent per droplet of thedeposition system. In even further embodiments, the droplet-on-demanddispensing system is able to dispense between about 1 pL to about 50 pLof reagent per droplet of the droplet-on-demand dispensing system.

In some embodiments, the reagents are dispensed from thedroplet-on-demand dispensing system and/or deposited at a velocity ofbetween about 0.5 m/s to about 20 m/s. In other embodiments, thereagents are dispensed from the device and/or deposited at a velocity ofbetween about 4 m/s to about 10 m/s.

As described further herein, different reagents or compositionscomprising reagents may be dispensed from the droplet-on-demanddispensing system. The skilled artisan will appreciate that differentreagent compositions or formulations may comprise different propertiesand, in some embodiments, may be dispensed at different shear rates. Byway of example, a primary stain reagent composition (or, for thatmatter, any small molecule dye) may be dispensed at a shear rate ofbetween about 1×10⁵ s⁻¹ and about 1×10⁷ s⁻¹. As another example, a largemolecule reagent composition may be dispensed at a shear rate less thanabout 2×10⁶ s⁻¹. As a further example, an antibody reagent solution maybe dispensed at a shear rate less than about 5×10⁵ s⁻¹. Appropriateshear rates may be determined for each composition by those of ordinaryskill in the art and the dispensing device may be tuned accordingly.

Applicants submit that the droplet-on-demand dispensing system of thepresent disclosure allows for precise dispensing of reagents onto abiological sample. Indeed, and as compared to the prior art, the amountor mass of reagent deposited onto the biological sample using thedisclosed dispensing device may be varied by “dialing-in” an amount ofreagent. The skilled artisan will recognize that an intensity of a stainmay thus be varied based on a particular sample and/or assay. Indeed,Applicants have surprisingly discovered that reagent mass may be variedby one of several methods including (i) applying reagent by multiplepasses of the dispensing mechanism, such as to provide a cumulativedeposition of reagent material (e.g. from 1 to about 25 passes or more);(ii) varying the dots per inch (dpi) of reagent dispensing (e.g. fromabout 50 dpi to about 1200 dpi); (iii) varying the droplet volume (e.g.from about 1 pl to about 1 nL); and/or (iv) varying the reagentconcentration in any reagent composition or formulation.

The present reagent dispensing device is believed to allow for lessreagent to be utilized and/or wasted as compared with prior arttechniques. Table 1 sets forth various staining processes andcomparatively illustrates slide coverage volume per assay step andspatial staining capability between different instruments. Table 1 setsforth reagent volume savings and the ability to stain specific regionsof a microscope slide using inkjet or another droplet generationtechnology in the context of an automated staining tissue stainingplatform. For assay steps, such as the droplet-on-demand systemdescribed herein, the total volumes required to cover an entire samplevary from about ten microliters to about less than a microliter. At thesame time, with a single-droplet volume of about ten Pico liters, it isbelieved that staining specific regions of about ten cells or less ispossible. By way of example, and as enumerated in Table 1, as comparedwith the prior art “dip and dunk” technique which requires about 10 mLto 100 mL of reagent per slide per assay step, the present device onlyrequires, in some embodiments, about 0.001 mL of reagent to be used perslide per assay step, resulting in a significant reduction in the volumeof reagent utilized (i.e. several orders of magnitude difference).Moreover, Applicants have discovered that the device according to thepresent disclosure allows for reaction kinetics to increase whencompared with prior art methods, as described further herein.

TABLE 1 Comparison of difference staining processes. Slide CoverageStaining Example Volume Spatial Staining Process Instrument per AssayStep Capability Dip and Dako Autostainer >10 mL per No Dunk LINK 48container est. Puddle VENTANA HE 600 ~1 mL est. No system MicrofluidicLeica BOND-III ~100 μL est. No Droplet Inkjet Stainer <1 to 10 uL Yes,10 cell Based (disclosed herein) regions est.

In the context of the primary stains hematoxylin and eosin, as the massof reagent dispensed increases (as the number of passes increased), theintensity of the stain increases (see, for example, FIGS. 4A to 4E and5A to 5C). FIGS. 4A to 4E illustrate hematoxylin reagent dispensing ontotonsil tissue fixed on a specimen slide (see Example 6 herein forstaining procedures). FIG. 4A qualitatively shows that with anincreasing number of passes, and increasing volume of reagent dispensed(4 μL/in², 8 μL/in², 14 μL/in²), the staining intensity increases forthe inkjet process. FIG. 4B illustrates the absorbance of thehematoxylin primary stain based on the total stain deposition (600 dpi,3 pL drop size, about 1 uL/in² per pass of the print head). Forcomparison with conventional staining apparatuses, absorbance valuesfrom control slides are shown in the indicated band of FIG. 4B. About 14μL/in² of stain deposition from the reagent dispensing device results ina stain approximately equivalent to that provided with conventionalstaining apparatus with a two (2) minute incubation time. As an exampleof staining according to the disclosed methods, FIG. 4C shows crispstaining of lymphoid follicles surrounding germinal centers, a criticalmacroscopic feature of quality tissue staining, as produced by theinkjet staining process described in this disclosure. In FIG. 4C, thearrow denotes a germinal center with strong staining in the lymphoidfollicle rim. FIG. 4D provides a microscopic view of the inkjet stainedtonsil tissue and shows distinct nuclear staining, including the abilityto see condensed chromatin and nuclear membranes in some nuclei. In FIG.4D the arrow denotes an example nucleus where condensed nuclear materialis visibly darker stained than the surrounding nucleus. FIG. 4E providesa view of a stained tonsil section demonstrating that the disclosedtechnique provides specific staining; in the field both stained nucleiand unstained red blood cells (denoted by the arrow) are present.

The experimental results illustrated in FIGS. 4A to 4E were obtainedusing a custom retrofitted EPSON C88+ printer with Non-OEM refillableink cartridges filled with a modified, commercially availablehematoxylin. The print characteristics were as described above. Inbrief, the experiment consisted of: 1) preparing 4 μm tonsil sectionfrom FFPE blocks, 2) preparing the section for staining, 3) creating aprint image file to instruct the printer on the reagent to be printed,the x-y coordinates for staining, and the pattern to be printed, 4)loading the tissue section and glass slide onto the print feedmechanism, 5) repeating the printing process to achieve the desiredstain intensity, and 6) manually washing off excess stain andcoverslipping the slide. Slides were imaged using a VENTANA iScan HTSlide Scanner and intensity values were extracted using ImageJ andMATLAB.

Without wishing to be bound by any particular theory, it is believedthat the significance of the results above is two-fold. First, itdemonstrates that “dialing-in” staining intensity (mass-limitedstaining) is enabled by the droplet-on-demand printing process (e.g.inkjet deposition or another small droplet deposition technology).Previously, this has not been a capability of any other tissue stainingtechnology, as all other technologies required a combination ofincubation time, temperature, or variable concentration reagents to“dial-in” the intensity of staining Second, the result demonstrates thatin this low-volume staining format, tissue dry-out does not result innon-specific staining, as demonstrated by the macroscopic andmicroscopic features visible in FIGS. 4C, 4D, and 4E.

By way of another example, FIGS. 5A to 5C illustrate eosin reagentdispensing onto colon tissue fixed on specimen slides. FIG. 5Aillustrates increasing eosin stain intensities with increasing staindeposition (5 μL/in², 15 μL/in², 25 μL/in²) utilizing the currentlydisclosed dispensing device and process. FIG. 5B illustrates theabsorbance of the eosin primary stain based on the total staindeposition. For comparison with conventional staining apparatuses,absorbance values from control slides are shown in the shaded band. Atabout 25 μL/in2 of stain deposition from the reagent dispensing deviceresults in an equivalent stain to a two (2) minute incubation time usinga conventional staining apparatus. FIG. 5C compares staining of colontissue using an inkjet process where three distinct shades of eosin canbe clearly observed, and this allows the skilled artisan todifferentiate tissue regions (smooth muscles, connective tissue, and redblood cells). In FIG. 5C, the reverse hatched arrow denotes a lightlystained region of connective tissue, the forward hatched arrow denotesan intensely stained region of red blood cells, and the white arrowdenotes the moderately stained smooth muscle surrounding a blood vessel.This experimental result was obtained using the procedure describedherein with the exception that hematoxylin was substituted for amodified eosin formulation. As in FIGS. 4A to 4E, FIGS. 5A to 5Cdemonstrate that “dialing-in” stain intensity is enabled by adroplet-on-demand reagent dispensing process and further that a lowvolume staining environment does not inhibit the required specificity ofstaining, as evidenced in FIG. 5C.

Of course, the skilled artisan will appreciate that stains may bedeposited in a multi-step process, e.g. where two primary stains (or anytwo reagents) are deposited onto a biological sample. FIGS. 6A and 6Bprovide an illustration of staining with both hematoxylin and eosinprimary stains using a droplet-on-demand dispensing device according tothe present disclosure, where the total stain volumes deposited wereabout 8 μL/in² and about 12 μL/in². Applicants have discovered thatmultiple dispensing staining operations do not interfere and that customreagents formulated for inkjet technology provide acceptable stainingresults. FIG. 6A shows the macroscopic staining result, with multiplemorphological regions of the tissue stained with the correspondinglydifferent ratios of hematoxylin and eosin, based on the microscopicstructure of that feature. FIG. 6B show a magnified view that shows highdegrees of nuclear staining on the lymphoid follicles of the germinalcenters and a high degree of eosin staining in the connective tissuebetween the active regions of this tonsil specimen. In FIG. 6B, the leftarrow denotes a region of a lymphoid follicle (darker hematoxylinstaining) surrounding a germinal center (lighter hematoxylin staining)and the upright arrow denotes a region of squamous epithelium, intenselystained with eosin.

The experimental result of FIGS. 6A and 6B were obtained using theDimatix DMP-2831 Materials Printer with the Dimatix DMC-11610 printcartridge. The drop size from these 16 nozzle print heads were known tobe fixed at about 10 pL and the droplet spacing for the print job wasset at about 1270 dpi for the printing of both hematoxylin and eosin.For this stain, custom inkjet formulations for hematoxylin and eosinwere developed, as described herein. As noted further herein, theseformulations were designed to achieve several objectives including: 1)improved dispensing reliability and consistency through the design ofthe physical characteristics of the fluids (i.e. setting the appropriateviscosity and surface tension for jet formation and droplet breakoff),2) improved stability of reagents in the cartridge (e.g. theintroduction of Aluminum Chloride to the hematoxylin formulation), and3) increasing the staining intensity of the formulations to minimize thenumber of printing passes required (e.g. darker eosin staining isproduced with five passes of the “inkjet eosin” as opposed to the 25passes required for the darkest stain in FIGS. 5A to 5C). The printingprocesses in this experiment consisted of the following steps: 1)creating a print image file for the position, shape, and size of thetissue section mounted on a glass slide, 2) loading a cartridge andprint head for the specific reagent to be printed onto the tissue, 3)setting the sabre angle of the print head to dial in the correct DPI forthe staining process, 4) loading the microscope slide and sample ontothe movement stage, and 5) executing the print job the desired number ofinteraction to evolve the proper staining intensity. The application ofbluing solution for hematoxylin or a solution to differentiate the eosinwas performed manually, offline.

In some embodiments, the reagent deposition device is configured toenable any reagent dispensed to penetrate a thin boundary layer of fluidand replenish staining reagents in communication with the sample.Without wishing to be bound by any particular theory, it is believedthat current staining technology relies upon puddles of stainingreagents which passively diffuse down a concentration gradient into thetissue sample. In these staining systems, which are believed to lackactive mixing of the reagent at the puddle-tissue interface, staindiffusion into tissue is mediated by the buildup of a stainconcentration depletion layer at the interface, limiting stainingkinetics. The present disclosure is believed to improve upon prior artstaining techniques by (i) creating staining films of a thicknessapproaching that of the depletion layer; and (ii) replenishing stainmolecules in the depletion layer, thereby overcoming the limitations ofpassive stain diffusion.

In some embodiments, the reagent, or composition comprising the reagent,is dispensed through an immiscible fluid with sufficient velocity todrive droplets of reagent through a thin film of tissue-preserving fluidmedium. Examples of thin film fluids include, but are not limited to,draksol, linpar, mineral oil, or silicone oil. Generally, favorableattributes include a liquid state at room temperature (e.g. 20-30° C.)low surface tension, and low vapor pressure. The liquid state of theimmiscible barrier layer allows for the resupply of aqueous fluidsthrough the barrier. The low surface tension allows for the barrier tobe coated onto the sample as a relatively thin film (100 μm in height orless). The low vapor pressure ensures that the barrier layer will beslow to evaporate off of the sample. It is believed that this drives thereagent into a layer in communication below the immiscible fluid. Forthis embodiment, the kinetic energy (a product of the mass of thedroplet and the impact velocity when the droplet hits the film) of thedroplet should be greater than the surface tension/energy of theprotective layer (plus, provide sufficient additional energy to accountfor displaced fluid), e.g. great than about 9.52×10⁻¹⁰ J. In someembodiments, the kinetic energy is about 6.23×10⁻¹⁰ J. Moreover, theWeber number of the droplet must be less than about 18 to ensure thatdroplet breakup does not occur on impact. In some embodiments, thedroplet must have a higher density than the protective film to ensurethat once the surface is broken, the droplet will continue through theprotective layer to contact tissue directly.

In other embodiments, the reagent is dispensed into a pre-existingaqueous fluid “puddle” with sufficient velocity that droplets of thereagent are driven into a thin film that will carry stain locallythrough the puddle to a fluid-tissue stain depletion layer. It isbelieved that this will facilitate the replenishment of reagent at theinterfacial contact point in communication with the sample. In turn, itis believed that this will eliminate the stain depletion boundary layerand improve the staining reaction kinetics which, in some cases, ismediated by the diffusion of staining reagent across the depletionlayer. Indeed, for large biomolecules, such as antibodies, binding ofthe molecule to a target is driven by time and concentration. Bycontinually disrupting the thin film with additional reagent materialvia dispensing with the presently disclosed device (and inherentmixing), the effective concentration at the tissue surface is enhanced,and believed to provide for faster uptake. For this embodiment, thevelocity generally ranges from about 5 m/s to about 15 m/s.

With reference to FIG. 7, an incoming slide containing a tissue sampleis first received by the device 110. In some embodiments, the tissuesample contains a protective fluid layer in communication with thesample to prevent the sample from drying out. Reagent is then dispensedonto the tissue section 120. Step 120 may be repeated as many times asnecessary to build stain intensity. In some embodiments, the reagent isoptionally incubated 130. For example, for large biomolecules, bindingmay be driven by time and concentration. Dispensing with or withoutincubation may be repeated 135 as necessary. Without wishing to be boundby any particular theory, it is believed that by continually disruptingthe thin film with addition dispensed reagent, the effectiveconcentration at the tissue surface is enhanced, resulting in fasteruptake. Following dispensing, remaining reagent and/or the protectivefilm is removed at step 140. In some embodiments, removing reagentand/or the protective film is facilitated by dispensing a wash solutionto dilute, increase surface tension, and/or to decrease viscosity. Thetissue section is then provided at step 150 for further downstreamprocessing or analysis.

FIG. 8A sets forth a further process map illustrating a process forimmunohistochemical staining in an automated fashion using inkjet orother droplet-on-demand technology. At step 701, the incoming sample mayinclude a deparaffinized tissue section, a frozen tissue section, or acell sample. Step 702 provides a general sample preparation step(s) tobridge from any previous process steps. Generally, this step(s) isdesigned to ensure that a consistent low volume of residual fluid ispresent within the sample and that the reaction conditions (e.g. bufferstrength, pH) are set appropriately for a subsequent inkjet ordroplet-based staining step. The dispense operation at step 702 may be adroplet based process or a bulk fluid dispense onto the sample with avolume from one microliter up to one milliliter. An optional fluidremoval portion is designed to precisely reduce the residual volume offluid remaining on the sample, to a final volume in the range of about100 nL to about 100 μL. Step 703 sets forth the introduction of atargeted protein binder to the tissue via an inkjet or droplet dispensetechnology, which encompasses individual droplet size from about 1 pL toabout 100 nL, print patterns as small as a single droplet up to the sizeof a standard microscope slide, and print densities from about 100 dpito about 1300 dpi.

Step 704 sets forth the optional practice of simultaneouslyco-dispensing another reagent with the reagent of interest for theparticular assay step. For small droplets, it is believed that thisfacilitates near-instant, on-slide mixing of reagents and imparts theunique ability to mix reagents in-situ to trigger reactions;ratiometrically dilute the concentration of biomolecules, dyes, orsubstrates; or homogenize the staining field across a sample. Step 705sets forth the period over which specific binding interactions occurbetween the dispensed reagent and the target sample. Step 706 sets forththe effects occurring to the deposited print droplets onto the tissuewhereby the small amount of active binder is consumed via reaction withthe tissue while the excess fluid is either actively or passivelyremoved from the sample. Since it is believed that these small volumesare thinner than the expected diffusion depletion layer observed inpuddle staining technology, these reactions are believed to proceedquickly and as a result may be replenished with additional activebinders to continue to rapidly drive the reaction toward the desiredendpoint or equilibrium.

Step 707 sets forth the means by which additional active binders are(biomolecules or dyes) are resupplied to the sample in order to continuedriving an efficient reaction. In this case the necessary print densityfor re-supply may be much smaller than the original print application.In some embodiments, a droplet density of between about 50 to about 100dpi may be sufficient to drive the reaction, while in other embodimentsa complete re-print of the original print density may be required.Finally, in step 708 the sample has completed this assay block and isready to move on to the next assay block, which may be a repeat of thisprocess but with different reagents and reaction conditions or may be adifferent process, such as automated coverslipping or imaging as part ofan integrated digital pathology workflow.

FIG. 8B provides an example result from staining according to theprocess of FIG. 8A. This example illustrates the result of thedeposition of an anti-CD20 primary antibody onto a FFPE section oftonsil, sectioned at 4 μm. The inkjet reagent used in this particularexample is described further herein. Generally, the process isapplicable for primary antibodies, secondary antibodies, linkers forenzyme mediated detection, enzymes for driving detection reactions, orsubstrate to the specifically linked enzyme. The process described hereis a non-obvious extension of the theory behind “Cyclicdraining-replenishing” technology, as described by Li, et al. (Small,2016, 12, No. 8, 1035-1043). In fact, the process described herein isbelieved to overcome some limitations of the process described by Li inthe sense that the resupply of reagent is directed with a non-zerovelocity into the tissue via an inkjet print head (approximately 8 m/sin this example). Further, in the work by Li, the focus was on acircular mixing process while in the present disclosure either an active(e.g. air flow) or passive (e.g. evaporation) process is used to drainthe excess fluid while the dye or biological is resupplied viaadditional print passes with the inkjet head (see step 707 of FIG. 8A).This in turn is believed to drive the reaction speed from adiffusion-mediated process (slow, characteristic of puddle-basedstaining technologies) to a binding kinetics mediated process (fast,unique to droplet deposition and film-based staining techniques wherethe depletion layer can be actively resupplies with biomolecules ordye).

FIGS. 9A, 9B, and 9C further illustrate the staining achieved throughthe deposition of primary antibodies using the droplet-on-demand systemand methods disclosed herein. In general, FIGS. 9A, 9B, and 9C set forthexamples of primary antibodies deposited using a droplet-on-demandprocess, using anti CD-20 antibodies on tonsil tissue. FIG. 9A comparesstaining intensity for a static thin film puddle versus a mixed thinfilm puddle. In this case, despite a lower total antibody exposure(μL×time), the thin film mixed via jetting of fluid into the filmresulted in a higher stain intensity. Details of the mechanism drivingthese differential results are illustrated in FIG. 8A. FIGS. 9B and 9Cshow regions of tonsil tissue stained according to the processes stainedherein, where for FIG. 9B the parameters are as follows: 14.4 μL/in²(1200 dpi, 10 pL drop, single print head pass), static film, 16-minuteincubation time. For FIG. 9C the parameters are as follows: 3.6 uL/in²(600 dpi, 10 pL drop size, single print head pass), four (4) minuteincubation time, mixing via the addition of 400 nL/in² (200 dpi, 10 pLdrop size, single print head pass) of antibody to the thin film thenanother four (4) minute incubation, followed by two (2) additionalmixing steps.

FIGS. 10A through 10C provide additional examples of the deposition ofprimary antibodies deposited using a droplet-on-demand reagentdeposition process according to the present disclosure (anti-CD20,tonsil tissue). FIGS. 10A to 10C further affirm that inkjet or dropletdeposition technologies provide sufficiently small fluid volumes suchthat binding kinetics are driven by the mass of reagent deposited ontothe target. FIG. 10A compares staining intensity for thin film puddlesof two different volumes to demonstrate that antibody binding for theinkjet thin film process is mediated by the amount of antibody deposited(i.e. CD20 signal strength increases as a function of the amount ofprimary antibody deposited on the tissue). FIGS. 10B and 10C are regionsof tonsil stained with the respective processes from FIG. 10A. Briefly,for FIG. 10B, 14.4 μL/in² (1200 dpi, 10 pL drop, single print head pass)of reagent was patterned as a static film onto the tissue section, andallowed to incubate at room temperature for 16 minutes. For FIG. 10C,28.8 μL/in² (1200 dpi, 10 pL drop, two (2) print head passes) of reagentwas patterned as a static film onto the tissue section, and allowed toincubate at room temperature for 16 minutes. Antibody formulations usedto prepare FIGS. 10A through 10C are described further herein.

In another aspect of the present disclosure, Applicants have found thatthe dispensing device of the present disclosure allows for x-y spatialcontrol of reagent deposition, such that tissue regions of interest maybe identified and stain selectively applied to those regions of abiological sample. In some embodiments, the dispensing device describedherein is combined with an imaging system such that particular regionsor cells within a sample may be treated with a reagent. In anotheraspect of the present disclosure is a method of applying at least onereagent to a specific region of a tissue specimen comprising the stepsof (a) imaging a tissue sample; (b) choosing a specific region of thetissue for application of the reagent; and (c) depositing the reagent tothe specific region of the tissue with a piezoelectric depositionsystem. In some embodiments, between about 360 nL/in² (600 dpi, 1pL/drop) to about 14.4 μL/in² (1200 dpi, 10 pL/drop) of reagent isapplied to the specific region of the tissue per pass of the depositionsystem.

For example, FIG. 11 illustrates a “tissue panel on a slide” embodimentwhich is enabled specifically by the spatial deposition/multiplexingmade possible using the disclosed inkjet staining process. Withreference to FIG. 11, antibodies for the IHC stains (labeled IHC1 andIHC4) are incubated on adjacent tissue sections simultaneously. At thesame time, a primary stain (labeled H&E) is prepared on another regionof the slides. After incubation with primary antibodies, the same ordifferent detection chemistry may be used on the IHC tissue sectionssince the primary antibodies are spatially separated onto differenttissue sections on the slide. It is believed that this would facilitatea quicker assay turnaround time as well as the convenience of a completediagnostic panel on a single slide. In some embodiments, again withreference to FIG. 11, a three (3) IHC marker breast panel may be run ona single slide, facilitating a pathologist workflow from themorphological staining with a primary stain through each of threediagnostic IHC markers for phenotyping breast cancer (e.g. HER2/neu,Estrogen Receptor, Progesterone Receptor).

Reagent Compositions

Overview

The skilled artisan will appreciate that any type of reagent or reagentcomposition may be dispensed using the reagent deposition device andprocess described herein. For example, in some embodiments, the reagentdispensed from the dispensing device is a primary stain, such ashematoxylin or eosin. In other embodiments, the reagent dispensed fromthe device is an antibody useful in histochemistry (e.g. primary andsecondary antibodies), a composition comprising an antibody or antibodyconjugate (e.g. enzyme conjugated antibodies, or an antibody conjugatedto a fluorophore, hapten, or other label), and/or detection reagents fordetecting an antibody or antibody-target complex (e.g. a compositioncomprising chromogenic substrates, secondary antibodies specific for alabel conjugated to a primary antibody, etc.).

In some embodiments, the reagents or compositions comprising reagentsare modified as compared with off-the-shelf reagents or compositionscomprising those reagents so as to better facilitate delivery anddispensing through an inkjet deposition apparatus or piezoelectricdeposition apparatus. For example, the reagents or compositionscomprising the reagents may be altered to have a certain density, pH,viscosity, or rheology. In some embodiments, any reagent composition maycomprise one or more of buffers, rheology modifiers, surfactants,carrier proteins, stabilizers, viscosity modifiers, humectants,preservatives, and other additives. The skilled artisan will be able toselect appropriate components in appropriate amounts to provide areagent composition having desirable properties so as to effectuatedispensing with inkjet technology.

In some embodiments, the reagent compositions of the present disclosurehave a rheology, i.e. a “flow” of the solution, to facilitate a singledroplet of reagent under one unit of excitation of piezo-membrane.Indeed, the reagent solutions of the present disclosure have beendeveloped such that they (i) allow for proper staining, (ii) are able toform stable thin films; and (iii) are able to be dispersed viapiezoelectric deposition. In some embodiments, the reagent compositionshave a density that is greater than about 1 g/mL. In other embodiments,the reagent compositions have a density that is between about 0.75 g/mLand about 1.5 g/mL.

In some embodiments, the viscosity of the reagent composition rangesfrom about 1 cp to about 40 cp. In other embodiments, the viscosity ofthe reagent composition ranges from about 4 cp to about 15 cp. In yetother embodiments, the viscosity of the reagent composition ranges fromabout 6 cp to about 10 cp. In some embodiments, the surface tension ofthe reagent composition ranges from about 20 dyne/cm to about 70dyne/cm. In other embodiments, the surface tension of the reagentcomposition ranges from about 20 dyne/cm to about 45 dyne/cm. In yetother embodiments, the surface tension of the reagent composition rangesfrom about 20 dyne/cm to about 35 dyne/cm

In general, the viscosity modifier for use in any of the reagentcompositions is selected from glycols such as ethylene glycols,diethylene glycol, polyethylene glycols, propylene glycols, dipropyleneglycols, glycol ethers, glycol ether acetates; saccharides andpolysaccharides such as guar gum, xanthan gum; celluloses and modifiedcelluloses such as hydroxy methylcellulose, methylcellulose, ethylcellulose, propyl methylcellulose, methoxy cellulose, methoxymethylcellulose, methoxy propyl methylcellulose, hydroxy propylmethylcellulose, carboxy methylcellulose, hydroxy ethylcellulose, ethylhydroxyl ethylcellulose, cellulose ether, cellulose ethyl ether, andchitosan.

In some embodiments, the compositions of the present disclosure may alsocomprise one or more low-volatile water soluble humectants.Representative examples of humectants include: (1) triols, such as;glycerol, 1,2,6-hexanetriol, 2-ethyl-2-hydroxymethyl-propane diol,trimethylolpropane, alkoxlated triols, alkoxylated pentaerythritols,saccharides, and sugar alcohols; and (2) diols, such as ethylene glycol,diethylene glycol, triethylene glycol, propylene glycol, polyalkyleneglycols having four or more alkylene oxide groups, 1,3-propane dial,1,2-butane diol, 1,3-butane diol, 1,4-butane diol, 1,2-pentane diol,1,5-pentanediol, 1,2-hexanediol, 1,6-hexane diol,2-methyl-2,4-pentanediol, 1,2-heptane diol, 1,7-hexane diol,2-ethyl-1,3-hexane diol, 1,2-octane diol, 2,2,4-trimethyl-1,3-pentanediol, 1,8-octane diol; and thioglycol or a mixture thereof. Desirablehumectants are polyhydric alcohols.

In some embodiments, the reagent compositions comprise one or morestabilizers. In general, the stabilizer may be selected from sodiumiodate, aluminum chloride hexahydrate, aluminum sulfate hexadecahydrate,and protein stabilizers (e.g. trehalose, glycerol, Globulins, BSA,etc.). It is believed that the inclusion of one or more stabilizers mayprevent the precipitation of reagent molecules. For example,hematoxylin, which is known in the art to precipitate out of solution,may be formulated with one or more stabilizers to mitigate or preventprecipitation out of solution, and thus avoid the clogging of reagentlines or print/inkjet dispensing heads.

In some embodiments, the surface tension modifier is a surfactant. Thesurfactant may be one of an anionic surfactant, a cationic surfactant, anon-ionic surfactant, or mixtures thereof. In some embodiments, anappropriate surfactant is selected such that (i) when combined with theother reagent components, it allows for the desired surface tension tobe achieved; (ii) does not denature proteins or other reagentcomponents; and/or (iii) it provides a low foam height.

Anionic surfactants are generally based upon sulfates, sulfonates,phosphates, or carboxylates and contain a water-soluble cation. Arepresentative formula of a sulfonate is R—SO3M where R is a hydrocarbongroup of from about 5 to 22 carbon atoms which may be linked through analkoxy or oxyalkoxy to the sulfonate functionality and M is awater-soluble cation such as an alkali metal. Anionic surfactantsinclude alkyl ether sulfates, alkyl sulfates and sulfonates, alkylcarboxylates, alkyl phenyl ether sulfates, sodium salts of alkylpoly(oxyethylene) sulfonates, sodium salts of alkyl benzyl sulfonate,such as sodium salts of dodecylbenzyl sulfonate and sodium lauryl ethersulfate. Anionic surfactants also include anionic phosphate esters.

In some embodiments, the surfactants include, but are not limited topolyoxyethylene alkyl ether, wherein the alkyl is (CH₂)_(M) and theoxyethylene is (C₂H₄O)_(N), wherein M is an integer from 5 to 16, from 8to 14, or from 10 to 12 and N is an integer from 10 to 40, from 15 to30, or from 20 to 28. In one embodiment, the surfactant ispolyoxyethylene lauryl ether having a formula (C₂H₄O)₂₃C₁₂H₂₅OH. Inanother embodiment, the surfactant is a polyoxyethylene (20) sorbitanmonoalkylate, the monoalkylate comprising between 8 and 14 carbons. Inanother embodiment, the surfactant is a linear secondary alcoholpolyoxyethylene having a formula C₁₂-₁₄H₂₅₋₂₉O(CH₂CH₂O]_(x), wherein xequals an integer between 2 and 12. In yet another embodiment, thesurfactant is a polyoxyethylene octyl phenyl ether. Exemplarysurfactants are sold under the names: Brij® 35, TWEEN®, Tergitol™,Triton™, Ecosurf™, Dowfax™, polysorbate 80™, BigCHAP, Deoxy BigCHAP,IGEPAL®, Saponin, Thesit®, Nonidet®, Pluronic F-68, digitonin,deoxycholate, and the like. Particular disclosed working embodimentsconcern using surfactants selected from Brij® 35, TWEEN®, Tergitol™,Triton™.

Cationic surfactants useful in compositions of the present disclosurecontain amino or quaternary ammonium moieties. Cationic surfactantsamong those useful herein are disclosed in the following documents: M.C.Publishing Co., McCutcheon's, Detergents & Emulsifiers, (North Americanedition 1979); Schwartz, et al.; Surface Active Agents, Their Chemistryand Technology, New York: Interscience Publishers, 1949; U.S. Pat. No.3,155,591, Hilfer, issued Nov. 3, 1964; U.S. Pat. No. 3,929,678,Laughlin et al., issued Dec. 30, 1975; U.S. Pat. No. 3,959,461, Baileyet al., issued May 25, 1976; and U.S. Pat. No. 4,387,090, Bolich, Jr.,issued Jun. 7, 1983.

Among the quaternary ammonium-containing cationic surfactant materialsuseful herein are those of the general formula:

wherein R1-R4 are independently an aliphatic group of from about 1 toabout 22 carbon atoms or an aromatic, alkoxy, polyoxyalkylene,alkylamido, hydroxyalkyl, aryl or alkylaryl group having from about 1 toabout 22 carbon atoms; and X is a salt-forming anion such as thoseselected from halogen, (e.g. chloride, bromide), acetate, citrate,lactate, glycolate, phosphate nitrate, sulfate, and alkylsulfateradicals. The aliphatic groups may contain, in addition to carbon andhydrogen atoms, ether linkages, and other groups such as amino groups.The longer chain aliphatic groups, e.g., those of about 12 carbons, orhigher, can be saturated or unsaturated. Especially preferred aremono-long chain (e.g., mono C₁₂ to C₂₂, preferably C₁₂ to C₁₈, morepreferably C₁₆, aliphatic, preferably alkyl), di-short chain (e.g., C₁to C₃ alkyl, preferably C₁ to C₂ alkyl) quaternary ammonium salts.

Salts of primary, secondary and tertiary fatty amines are also suitablecationic surfactant materials. The alkyl groups of such aminespreferably have from about 12 to about 22 carbon atoms, and may besubstituted or unsubstituted. Such amines, useful herein, includestearamido propyl dimethyl amine, diethyl amino ethyl stearamide,dimethyl stearamine, dimethyl soyamine, soyamine, myristyl amine,tridecyl amine, ethyl stearylamine, N-tallowpropane diamine, ethoxylated(with 5 moles of ethylene oxide) stearylamine, dihydroxy ethylstearylamine, and arachidylbehenylamine Suitable amine salts include thehalogen, acetate, phosphate, nitrate, citrate, lactate, and alkylsulfate salts. Such salts include stearylamine hydrochloride, soyaminechloride, stearylamine formate, N-tallowpropane diamine dichloride,stearamidopropyl dimethylamine citrate, cetyl trimethyl ammoniumchloride and dicetyl diammonium chloride. Preferred for use in thecompositions herein are cetyl trimethyl ammonium chloride, stearyltrimethyl ammonium chloride, tetradecyltrimethly ammonium chloride,dicetyldimethyl ammonium chloride, dicocodimethyl ammonium chloride andmixtures thereof. More preferred is cetyl trimethyl ammonium chloride.

The compositions of the disclosure may also include various non-ionicsurfactants. Among the suitable nonionic surfactants are condensationproducts of C₈-C₃₀ alcohols with sugar or starch polymers. Thesecompounds can be represented by the formula (S)_(n)—O—R, wherein S is asugar moiety such as glucose, fructose, mannose, and galactose; n is aninteger of from about 1 to about 1000, and R is C₈-C₃₀ alkyl. Examplesof suitable C₈-C₃₀ alcohols from which the R group may be derivedinclude decyl alcohol, cetyl alcohol, stearyl alcohol, lauryl alcohol,myristyl alcohol, oleyl alcohol, and the like. Specific examples ofthese surfactants include decyl polyglucoside and lauryl polyglucoside.

Other suitable nonionic surfactants include the condensation products ofalkylene oxides with fatty acids (i.e., alkylene oxide esters of fattyacids). These materials have the general formula RCO(X)_(n)OH, wherein Ris a C₁₀-C₃₀ alkyl, X is —OCH₂CH₂— (derived from ethylene oxide) or—OCH₂CHCH₃— (derived from propylene oxide), and n is an integer fromabout 1 to about 200.

Yet other suitable nonionic surfactants are the condensation products ofalkylene oxides with fatty acids (i.e., alkylene oxide diesters of fattyacids) having the formula RCO(X)_(n)OOCR, wherein R is a C₁₀-C₃₀ alkyl,X is —OCH₂CH₂— (derived from ethylene oxide) or —OCH₂CHCH₃— (derivedfrom propylene oxide), and n is an integer from about 1 to about 200.Yet other nonionic surfactants are the condensation products of alkyleneoxides with fatty alcohols (i.e., alkylene oxide ethers of fattyalcohols) having the general formula R(X)_(n)OR′, wherein R is C₁₀-C₃₀alkyl, n is an integer from about 1 to about 200, and R′ is H or aC₁₀-C₃₀ alkyl.

Still other nonionic surfactants are the compounds having the formulaRCO(X)_(n)OR′ wherein R and R′ are C₁₀-C₃₀ alkyl, X is —OCH₂CH₂—(derived from ethylene oxide) or —OCH₂CHCH₃— (derived from propyleneoxide), and n is an integer from about 1 to about 200. Examples ofalkylene oxide-derived nonionic surfactants include ceteth-1, ceteth-2,ceteth-6, ceteth-10, ceteth-12, ceteraeth-2, ceteareth6, ceteareth-10,ceteareth-12, steareth-1, steareth-2, stearteth-6, steareth-10,steareth-12, PEG-2 stearate, PEG4 stearate, PEG6 stearate, PEG-10stearate, PEG-12 stearate, PEG-20 glyceryl stearate, PEG-80 glyceryltallowate, PPG-10 glyceryl stearate, PEG-30 glyceryl cocoate, PEG-80glyceryl cocoate, PEG-200 glyceryl tallowate, PEG-8 dilaurate, PEG-10distearate, and mixtures thereof. Still other useful nonionicsurfactants include polyhydroxy fatty acid amides disclosed, forexample, in U.S. Pat. Nos. 2,965,576, 2,703,798, and 1,985,424, whichare incorporated herein by reference.

Exemplary surfactants include Tomadol 1200 (Air Products), Tomadol 900(Air Products), Tomadol 91-8 (Air Products), Tomadol 1-9 (Air Products),Tergitol 15-S-9 (Sigma), Tergitol 15-S-12 (Sigma), Masurf NRW-N (PilotChemical), Bio-Soft N91-6 (Stepan), and Brij-35 (Polyethylene glycoldodecyl ether) (Sigma).

To demonstrate that the reaction conditions inherent to the tissue priorto the printing of staining fluids impact the stain quality for thedisclosed staining method, a systematic study of different tissue pH'sprior to the printing of hematoxylin was undertaken in 4 μm liversections mounted on microscope slides. The pH of the tissue section wasset by applying 300 μL of a buffer solution to the tissue and thenprinting hematoxylin and eosin onto the sample using the assay describedin Table 8 during the first “Apply Wash (pH wash optional)” step. FIG.12A compares the ratio of staining intensity in the nuclei to the stainintensity of the cytoplasmic and extracellular regions of tissue. It washypothesized that the best ratio of these values should represent theoptimal staining conditions and that for an increasing pH, overall stainintensity (both specific to the nuclei and non-specific hematoxylinstaining should increase). While it was true that staining intensityincreased overall as pH increased to a point, at the high end of thescale overall staining intensity decreased (FIG. 12B) to an unacceptablelevel for a pre-staining buffer with a pH of five. Therefore, the nextbest condition was selected, a pre-staining buffer with a pH of 3.5.This method could be extended to uniquely adjust the pre-staining tissuebuffering conditions for unique tissue types or different stainingchemistries and biochemistries.

Table 2 details the capability of a two print head system to deliver abuffering pH solution to a tissue. In one embodiment, the solutions tobe dispensed are a weak acid solution and dilute sodium hydroxide. Inthis case the pH of the dispensed film can be adjusted by adjusting theDPI of the print pattern for each of the fluids. This relates to FIG.12B where it can be seen that the pH of the tissue prior to printinghematoxylin has an effect on both the stain intensity and stainspecificity. This application of inkjet staining is appropriate to meetthis need with in-situ mixing the two solutions during print deposition.

TABLE 2 Ratiometric formulations of buffer systems to set the pH oftissue. Binary Print Binary Print Example Example Buffer Buffer Volumeratio print ratio Target Actual Component 1 Component 2 (vol 1:vol 2)(dpi 1:dpi 2) pH pH formic acid, NaOH, 31 mM 1:1 500 dpi:500 dpi 3 3.002189 mM formic acid, NaOH, 31 mM 1:2 460 dpi:650 dpi 3.5 3.496 189 mMformic acid, NaOH, 31 mM 1:3 370 dpi:640 dpi 4 4.086 189 mM acetic acid,NaOH, 38.8 mM 1:1 500 dpi:500 dpi 4 4.094 34.8 mM acetic acid, NaOH,38.8 mM 10:17 460 dpi:600 dpi 4.5 4.500 34.8 mM acetic acid, NaOH, 38.8mM 10:23 400 dpi:607dpi 5 5.083 34.8 mM

Primary Stain Reagent Compositions

In the context of a primary stain reagent composition, the compositioncomprises a dye, a stain, or a “primary stain” (as that term is definedhere), a viscosity modifying agent, and a surface tension modifyingagent. While certain embodiments or examples herein may refer to aprimary stain composition comprising hematoxylin or eosin, the skilledartisan will appreciate that primary stain reagent compositions are notlimited to these particular dyes and that other dyes, stains, “primarystains,” or agents that otherwise enhance the visible contrast ofbiological structures in a tissue sample may be formulated in a likemanner without limitation.

In some embodiments, the amount of viscosity modifying agent ranges fromabout 35% to about 60% by total weight of the primary stain reagentcomposition. In other embodiments, the amount of viscosity modifyingagent ranges from about 25% to about 75% by total weight of the primarystain reagent composition. In some embodiments, where a dissolved solidis included (e.g. PEG), the amount of viscosity modifying agent mayrange from about 2% to about 60% by total weight of the primary stainreagent composition. In other embodiments, where a dissolved solid isincluded, the amount of viscosity modifying agent may range from about0.1% to about 2% by total weight of the primary stain reagentcomposition.

In some embodiments, an amount of surface tension modifying agent rangesfrom about 0.01% to about 0.5% by total weight of the primary stainreagent composition. In other embodiments, an amount of surface tensionmodifying agent ranges from about 0.001% to about 1% by total weight ofthe primary stain reagent composition

In some embodiments, primary stain reagent composition has a viscosityof 1 cp to about 40 cp. In other embodiments, primary stain reagentcomposition has a viscosity of 6 cp to about 10 cp. In some embodiments,the primary stain reagent composition has a surface tension up to about70 dyne/cm. In other embodiments, the primary stain reagent compositionhas a surface tension of about 25 dyne/cm to about 45 dyne/cm.

In some embodiments, the primary stain reagent solution furthercomprises one or more stabilizers and/or buffering agents. In someembodiments, the stabilizers include Aluminum Chloride, AluminumSulfate. In some embodiments, the buffering agents include Acetate,carbonate, phosphate, Tris-HCl, acetic acid, tris buffer, and phosphatebuffer. In general, an amount of stabilizers included within any primarystain reagent composition ranges from about 1% to about 20% by totalweight of the primary reagent stain composition. Likewise, an amount ofbuffers included within any primary stain reagent composition rangesfrom about 0.5% to about 5% by total weight of the primary reagent staincomposition.

Large Molecule Reagent Compositions

In some embodiments, a large molecule reagent composition comprises abiological molecule (e.g. an antibody, an antibody conjugate, an enzyme,a multimer, etc.), a viscosity modifying agent, and a surface tensionmodifying agent. In some embodiments, the large molecule reagentcomposition further comprises one or more carrier proteins (e.g. bovineserum albumin, normal goat serum). In some embodiments, the largemolecule reagent composition further comprises a buffering agent and/ora preservative composition. In some embodiments, the large moleculereagent composition has a viscosity ranging up to about 15 cp. In otherembodiments, the large molecule reagent composition has a viscosityranging from about 4 cp to about 11 cp. In yet embodiments, the largemolecule reagent composition has a viscosity ranging from about 4 cp toabout 7 cp. In some embodiments, the large molecule reagent compositionhas a surface tension ranging from about 20 dyne/cm to about 40 dyne/cm.In other embodiments, the large molecule reagent composition has asurface tension ranging from about 25 dyne/cm to about 35 dyne/cm.

In some embodiments, an amount of viscosity modifying agent ranges fromabout 1% to about 50% by total weight of the large molecule reagentcomposition. In other embodiments, an amount of viscosity modifyingagent ranges from about 25% to about 75% by total weight of the largemolecule reagent composition. In some embodiments, an amount of surfacetension modifying agent ranges from about 0.01% to about 0.5% by totalweight of the large molecule reagent composition. In other embodiments,an amount of surface tension modifying agent ranges up to about 1% bytotal weight of the large molecule reagent composition. The skilledartisan will recognize that any included carrier proteins and/or theprimary antibody itself may have an effect on surface tension and, insome embodiments, may contribute to a reduction of the surface tension.The skilled artisan will be able to take this factor into account whendetermining the quantity of any surface tension modifying agent forinclusion within the antibody reagent composition.

Non-limiting examples of antibodies which may be part of any largemolecule reagent composition include antibodies specific for cluster ofdifferentiation markers (e.g. CD20, CD3, CD4, CD8, CD45, CD25, CD163etc.), Ki-67, EGFR, HER2, HPV, ALK, BRAF, OX-40, PD-1, IDL-1, FoxP3, andCTLA-4.

Non-limiting examples of enzymes which may be part of any large moleculereagent composition include horseradish peroxidase, alkalinephosphatase, acid phosphatase, glucose oxidase, β-galactosidase,β-glucuronidase or β-lactamase.

EXAMPLES

The non-limiting examples which follow are intended to furtherillustrate certain embodiments of the present disclosure.

Example 1 Hematoxylin Formulation

TABLE 3 One embodiment of a hematoxylin formulation. Amount AmountComponent INGREDIENTS (g) (wt %) Description DI Water 152.3 57.07Propylene Glycol (~40% w/w) 101.6 38.07 Viscosity Modifier HematoxylinDye 2.27 0.85 Primary Stain Sodium Iodate 0.24 0.09 Oxidizer AluminumChloride Hexahydrate 2.56 0.96 Stabilizer Aluminum SulfateHexadecahydrate 6.67 2.50 Stabilizer Tomadol 900 (5 uL/mL, 0.98 g/mL)1.225 0.46 non-ionic surfactant 266.865 100.00

The composition of Example 1 was found to be sufficient for dispersionby the disclosed piezoelectric deposition method. The final pH of thecomposition was about 2.22; the surface tension was about 30 dyne/cm;and the viscosity was about 5 cp.

In the case of hematoxylin for inkjet dispensing, several mitigationswere discovered to improve the reliability and robustness of the dropletformation process for dispensing this particular stain. First, theinclusion of Aluminum Chloride in the formulation improved the overallstability of the formulation against spontaneous aggregation andprecipitation due to insolubility of long-chain metal-ion complexes.

Second, the large fraction of propylene glycol reduced dry out of thehematoxylin formulation when exposed to air by lowering the vaporpressure of the mixture as compared to formulations with a higher waterfraction. Both of these improvements represented non-standard (ornon-traditional) formulation characteristics targeted at creating ahematoxylin formulation uniquely suited to the inkjet form-factor.

Common to the field of functional printing with inkjet technology is theneed to flush significant volumes of ink through a print head to primethe system after use with another fluid. This stems from the design ofprint head systems fed through multiple ink reservoirs and the resultinglarge dead volumes. In some designs, this can account for greater than20% loss in ink. Likewise, the VENTANA HE 600 system has a sharedreagent manifold and during the purge/prime cycles, over 30% of thetotal assay volume is consumed. In the disclosed concept for an inkjetdispensing apparatus, Applicants utilized a cartridge-based inkjetsystem to overcome these limitations. By preparing complementaryformulations of reagents unique to and customized for the printingsystem, many sources of nozzle fouling (i.e. failure to dispensedroplets) were mitigated. However, it was also demonstrated that a primecycle of 5 μL or less per day was adequate to maintain reliable dispenseintegrity throughout the daily use of an inkjet reagent cartridge andover for the entire life of the reagent inkjet cartridge.

Example 2 Antibody Formulation

TABLE 4 One embodiment of an antibody formulation Amount ComponentINGREDIENTS (g) Amount (g) (% wt) Description Water 117.77 46.99%Glycerol 125 49.87% viscosity modifier Tris HCl 1.97 0.79% bufferProClin 300 (1.03 g/ml) 0.26 0.10% preservative Bovine Serum Albumin,2.5 1.00% carrier protein Frac. V Normal Goat Serum 2.5 1.00% carrierprotein Bio-Soft N91-8 (1.020 g/ml) 0.64 0.26% non-ionic surfactantSodium Hydroxide as needed Variable pH adjuster Primary Antibodyvariable Variable active staining component TOTAL 250.64 100.00%

The composition of Example 2 was found to be sufficient for dispersionby the disclosed piezoelectric deposition method. The surface tensionwas about 28 dyne/cm; and the viscosity was about 7 cp.

Example 3 Eosin Formulation

TABLE 5 One embodiment of an eosin formulation Amount Amount ComponentINGREDIENTS (g) (g) (% wt) Description DI Water 204.8 39.17% PropyleneGlycol (~60% w/w) 307.5 58.81% viscosity modifier Eosin Y 3.8143 0.73%dye Glacial Acetic Acid 5.5 1.05% pH adjuster Tomadol 900 (2.5 uL/mL,1.274 0.24% non-ionic 0.98 g/mL) surfactant 522.8883 100.00%

The composition of Example 3 was found to be sufficient for dispersionby the disclosed piezoelectric deposition method. The final pH of thecomposition was about 4.299; the surface tension was about 41 dyne/cm;the density was 1.042 g/mL; and the viscosity was about 8.1 cp.

Example 4 An Enzyme/Multimer Detection Formulation

Table 5 sets forth an embodiment of an enzyme/or multimer detectionformulation. In this particular non-limiting embodiment, the activestaining component is Mouse anti-hydroquinone horseradish peroxidase(anti-HQ HRP).

TABLE 6 One embodiment of an Enzyme/Multimer Detection FormulationAmount Amount Ingredients (g) (% wt) Component Description DeionizedWater 50 49.25% Propylene Glycol (1.04 g/ml) 50 49.25% ViscosityModifier Potassium Phosphate 0.8785 0.87% Buffer dibasic SodiumPhosphate 0.138 0.14% Buffer Monobasic Sodium Chloride 0.16 0.16% BufferLiquid Brij, 30% 0.086 0.08% Surfactant Goat Globulins 0.15 0.15%Carrier Protein B5 Blocker 0.068206 0.07% Blocker, non-specific bindingProClin 300 (1.03 g/ml) 0.02575 0.03% Preservative 6N HCl/6N NaOH asneeded as needed pH adjuster Mouse anti-HQ HRP 0.025 0.025% ActiveStaining Component (Enzyme- mediated detection) Total 101.531456 100.00%

Example 5 An Alternative Antibody Formulation with a Large MolecularWeight Viscosity Modifier

An alternative antibody formulation in accordance with the presentdisclosure is set forth in Table 7.

TABLE 7 An alternative antibody formulation. Amount Amount ComponentIngredients (g) (% wt) Description Deionized Water 54.274 54.27%Glycerol 40 40.00% Viscosity Modifier Dextran (avg MW 450 kD) 2 2.00%Viscosity Modifier Tris HCl 0.95 0.95% Buffer Bovine Serum Albumin, 1.21.20% Carrier Protein Frac. V Normal Goat Serum 1.2 1.20% CarrierProtein Bio-Soft N91-8 (1.020 g/ml) 0.256 0.26% Surfactant ProClin 300(1.03 g/ml) 0.12 0.12% Preservative 6N NaOH as as needed pH adjusterneeded Primary Antibody variable variable Active Staining ComponentTotal 100 100.00%

Example 6 Comparison of a Traditional Staining Procedure to an InkjetDeposition Staining Procedure

Set forth herein is a comparison of example assays from a conventionalsingle-slide staining (Table 9) system and the droplet-on-demanddispensing means described in this disclosure (Table 8). Both assaytables assume an offline deparaffinization process as well as a manualworkup to coverslipping after the assay is complete. While the VENTANAHE 600 assay for H&E staining (representing a conventional single slidestaining apparatus) may be “dialed-in” to adjust the staining usingincubation times only, the Inkjet Staining process offers severaladjustment point unique to a droplet deposition (i.e. inkjet) stainingprocess. First, staining is fundamentally mass-limited, as shown inFIGS. 4A and 5B, while a primary driver for stain intensity onconventional staining system is incubation time. In fact, this is theonly customer-facing feature to “dial” the assay intensity andspecificity in the example of a conventional staining apparatus.

TABLE 8 Inkjet Staining Assay Script Process Active Assay Volume FluidTime Time Step (μl) Process (min) (min) Notes Set Slide — — — Stainingintensity may be Temperature, adjusted using assay 40 C. temperature HW— 0.6 0 From slide “park” to pH wash Initialization (first location,includes height Z adjust for print heads) Apply pH 300 bulk, on 0 0Background/non-specific Wash tissue staining is mitigated by “setting”the tissue pH prior to printing hematoxylin Remove — 0.180 0.100 removesfluid down to ~5 uL Fluid Print    2.5 1000 × 1.580 1.580 Hematoxylin1000 print, 10 pL/drop Incubation — 1.000 1.000 Incubation steps areoptional to drive stain intensity Print    2.5 1000 × 1.580 1.580Hematoxylin 1000 print, 10 pL/drop Incubation — 1.000 1.000 Print    2.51000 × 1.580 1.580 Staining intensity may be Hematoxylin 1000 adjustedby printing onto the print, tissue multiple times 10 pL/drop Incubation— 1.000 1.000 Apply 300 bulk, on 0.033 0.033 Removes unbound Wash (pHtissue hematoxylin prior to bluing. wash May “clean up” backgroundoptional) staining Incubation — 1.000 1.000 Optional “soaking” step tohomogenize the stain across the tissue Remove 0.180 0.100 Fluid Apply300 bulk, on 0.033 0.033 Wash tissue Incubation — 1.000 1.000 Remove0.180 0.100 Fluid Apply 300 bulk, on 0.050 0.050 pH adjustment to basicBluing tissue conditions, locks the specific staining of hematoxylinonto the tissue Incubation — 0.500 0.500 Apply 300 bulk, on 0.130 0.033Remove excess bluing. Option Wash tissue to adjust pH to dial eosinintensity Remove 0.180 0.100 Fluid Apply 300 bulk, on 0.033 0.033 Wash(pH tissue wash optional) Remove 0.180 0.100 Fluid Print Eosin    2.51000 × 1.580 1.580 May be made ultra-bright by 1000 managing pH prior toprint, application. No incubation 10 pL/drop necessary Apply 300 bulk,on 0.033 0.033 Differentiation of eosin into Wash tissue three shades(RBCs, Remove 0.180 0.100 Fluid Apply 300 bulk, on 0.033 0.033 Removalof excess eosin Wash tissue Remove 0.180 0.100 Fluid Apply 300 bulk, on0.033 0.033 Wash tissue Remove 0.180 0.100 Fluid

However, on the inkjet staining system the primary drivers for dialingstain intensity are the number of print passes and the DPI (drop perinch) or density of the print area, both of which adjust the total massof staining material deposited. In FIG. 4A, the hematoxylin intensity isdriven up by increasing the number of print passes on the tissue andlikewise in FIG. 5A, the eosin intensity is similarly driven up by thedeposition of additional print layers on the tissue. As shown in Table8, for one embodiment of the disclosed staining process, the assay timeis fixed (due to the “dialability” of stain intensity with massdeposition), as opposed to variable for the conventional process (due tothe “dialability” of stain intensity being driven by incubation time).Further there is a significant reduction in the amount of liquid wastegenerated, from about 14.24 mL for the convention process down to about2.71 mL for an embodiment of the inkjet staining process. This resulthighlights the assay volume miniaturization effect possible with aninkjet deposition system, as suggested in Table 10.

TABLE 9 Conventional staining apparatus assay script Process VolumeFluid Time Assay Step (uL) Process (min) Notes Set Stainer Air — — Fixedtemperature control in Temperature, 45 C. conventional system Wash,Incubate, 1000.00 Bulk 0.333 Remove Fluid Wash, Incubate, 1000.00 Bulk0.333 Remove Fluid Hematoxylin, 1350.00 Bulk variable, Stain intensityis driven Incubate, Remove 1-10 using incubation time, which Fluid maybe between one and ten minutes Wash, Incubate, 1000.00 Bulk 0.667 RemoveFluid Acid Wash, Incubate, 1200.00 Bulk variable, Specific staining isdriven Remove Fluid 0-3 using acid wash incubation time, which isvariable and also decreases stain intensity Wash, Incubate, 900.00 Bulk0.333 Remove Fluid Bluing, Incubate, 1050.00 Bulk 0.500 Remove FluidWash, Incubate, 900.00 Bulk 0.333 Remove Fluid Eosin, Incubate, 1350.00Bulk variable, Remove Fluid 0.5-7 Wash, Incubate, 1000.00 Bulk 0.333Remove Fluid Wash 1000.00 Bulk 0.333 Wash 1000.00 Bulk 1.333 Purge/PrimeSteps 1870.61 — — Necessary to change to Between Fluids Total differentfluids on a (Hematoxylin) conventional system Purge/Prime Steps 2100.99— — Necessary to change to Between Fluids Total different fluids on a(Wash) conventional system Purge/Prime Steps 650.14 — — Necessary tochange to Between Fluids Total different fluids on a (Acid Wash)conventional system Purge/Prime Steps 579.99 — — Necessary to change toBetween Fluids Total different fluids on the (Bluing) conventionalsystem Purge/Prime Steps 610.78 — — Necessary to change to BetweenFluids Total different fluids on a (Eosin) conventional

Table 10 which follows illustrates an inkjet deposition processaccording to embodiments of the present disclosure. As compared with theVENTANA HE 600 process, for the inkjet staining system the primarydrivers for dialing stain intensity are the number of print passes andthe DPI (drop per inch) or density of the print area, both of whichadjust the total mass of staining material deposited. In this particularassay, the total assay volume utilized was about 2.71 mL, and the totalassay time was about 14.24 minutes.

TABLE 10 Summary comparison of inkjet and conventional assaysConventional Inkjet H&E H&E Total Staining Assay Time 6.00 to 24.5014.24 (min) Total Assay Volume (mL) 19.11 2.71

While a small incubation period is still a component of some processingsteps for staining via the disclosed inkjet deposition processes, theapplication of an intense eosin stain (for staining the cytoplasm) doesnot require any additional incubation after the printing. Withoutwishing to be bound by any particular theory, it is believed that thisillustrates that staining is mass-limited when using ultra-low volumesor reagent, as well as the fact that reaction kinetics may be improved(reduction of staining time from seven minutes to one minute) even witha 100-fold reduction in reagent, as compared with traditional stainingtechniques.

Example 7 Purging and Cleaning

Two common practices in the field of inkjet printing are purging andblotting a print head to induce fluid to flow into the capillary spaceand prime the nozzles for dispensing. Purging refers to the applicationof positive pressure within the ink container in order to force jets offluid out of the nozzles, without actuating the piezoelectric or thermaldroplet generation elements. Purging may also be used to removeocclusions (e.g. solid crystals, protein aggregates) and allow theactuation of droplets from the nozzle. Blotting refers to theapplication of a wicking pad to the outside of the nozzle area of theprint head. This induces flow through the nozzles to prime for printingor to clean up any residual fluid on the print head. Both these methodsare employed on the current inkjet staining system for managing “wellbehaved” fluids (i.e. those fluids without a tendency to crystallize andocclude the nozzles).

In investigating inkjet staining, several methods of cleaning andmaintaining print heads were discovered using physical or chemicaltreatment. Generally, physical treatment was the preferred method as itwas less destructive and may be automated relatively easily. For themaintenance of print heads, a moist, local high humidity environment wasa key factor in preventing crystallization or precipitate formation atthe nozzles. By resting a blotting pad filled with a mixture of waterand glycol against the nozzle plate during long-term storage on theinkjet staining system, dry out was mitigated.

Two effective solvent systems used for the cleaning of crystalizedmaterial from the print heads were 3% periodic acid and a mixture ofhydrogen peroxide/Sodium Carbonate. Both were effective at removingocclusions from the print nozzles.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet are incorporated herein by reference, intheir entirety. Aspects of the embodiments can be modified, if necessaryto employ concepts of the various patents, applications and publicationsto provide yet further embodiments.

Although the disclosure herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent disclosure. It is therefore understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present disclosure as defined by the appended claims.

1. A method of dispensing reagent onto a biological sample comprising:overlaying a protective fluid layer onto a biological sample, thebiological sample being disposed on a support medium; dispensing reagentdroplets of between about 1 pL to about 50 pL such that the reagentdroplets penetrate the protective fluid layer and contact the biologicalsample; wherein the reagent droplets comprise a reagent compositionselected from the group consisting of a primary stain reagentcomposition and an antibody reagent composition.
 2. The method of claim1, wherein the reagent droplets are dispensed at a velocity of betweenabout 5 m/s to about 15 m/s.
 3. The method of claim 1, wherein theprotective fluid layer is an aqueous puddle.
 4. The method of claim 1,wherein the protective fluid layer is an immiscible oil.
 5. The methodof claim 4, wherein a density of the reagent droplets is greater than adensity of the immiscible oil.
 6. The method of claim 1, wherein akinetic energy of the reagent droplets is greater than a surface tensionof the protective fluid layer.
 7. The method of claim 6, wherein thekinetic energy is greater than 9.52×10⁻¹⁰ Joules.
 8. The method of claim1, wherein the primary stain reagent composition comprises a dye, asurfactant, and a viscosity modifier, wherein the composition has aviscosity ranging from about 1 cp to about 40 cp and a surface tensionranging from about 25 dyne/cm to about 45 dyne/cm.
 9. The method ofclaim 8, wherein the dye is selected from the group consisting ofhematoxylin, eosin acridine orange, bismark brown, carmine, coomassieblue, cresyl violet, crystal violet, DAPI(“2-(4-Amidinophenyl)-1H-indole-6-carboxamidine”), ethidium bromide,acid fucsine, Hoechst stains, iodine, malachite green, methyl green,methylene blue, neutral red, nile blue, nile red, osmium tetraoxide,rhodamine, and safranine.
 10. The method of claim 8, wherein theviscosity of the primary stain reagent composition ranges from about 6cp to about 10 cp.
 11. The method of any of claims 8 to 10, wherein theprimary stain reagent solution is dispensed at a shear rate of betweenabout 1×10⁵ s⁻¹ and about 1×10⁷ s⁻¹.
 12. The method of claim 1, whereinthe antibody reagent composition comprises a primary antibody, asurfactant, and a viscosity modifier, wherein the composition has aviscosity ranging from about 4 cp to about 7 cp, and a surface tensionranging from about 20 dyne/cm to about 40 dyne/cm.
 13. The method ofclaim 12, wherein the antibody composition is dispensed at a shear rateof less than about 5×10⁵ s⁻¹.
 14. A method of dispensing reagent onto abiological sample comprising: overlaying a protective fluid layer onto abiological sample, the biological sample being disposed on a supportmedium; dispensing a pH modifier to the biological sample; anddispensing reagent droplets at a velocity of between about 5 m/s toabout 15 m/s; wherein the reagent droplets comprise a reagentcomposition selected from the group consisting of a primary stainreagent composition and an antibody reagent composition.
 15. The methodof claim 14, wherein an amount of reagent droplets dispensed ranges fromabout 10 μL/in² to about 30 μL/in².
 16. The method of claim 14, whereinthe pH modifier has a pH ranging from about 3 to about
 5. 17. The methodof claim 14, wherein the protective fluid layer is an immiscible oil andwherein a kinetic energy of the reagent droplets is greater than asurface tension of the immiscible oil.
 18. The method of claim 14,wherein the primary stain reagent composition comprises a dye, asurfactant, and a viscosity modifier, wherein the composition has aviscosity ranging from about 1 cp to about 40 cp and a surface tensionranging from about 25 dyne/cm to about 45 dyne/cm.
 19. The method ofclaim 18, wherein the primary stain reagent composition is dispensed ata shear rate of between about 1×10⁵ s-¹ and about 1×10⁷ s⁻¹.
 20. Themethod of claim 14, wherein the antibody reagent composition comprisinga primary antibody, a surfactant, and a viscosity modifier, wherein thecomposition has a viscosity ranging from about 4 cp to about 7 cp, and asurface tension ranging from about 20 dyne/cm to about 40 dyne/cm. 21.The method of claim 20, wherein the antibody composition is dispensed ata shear rate of less than about 5×10⁵ s⁻¹.
 22. A method of dispensingreagent onto a biological sample comprising: overlaying a protectivefluid layer onto a biological sample, the biological sample beingdisposed on a support medium; dispensing reagent droplets with a kineticenergy of greater than 9.52×10⁻¹⁰ Joules, and dispensing such that aspatial density of reagent droplets deposited on the biological sampleranges from about 50dpi to about 1200dpi, wherein the reagent dropletscomprise a reagent composition selected from the group consisting of aprimary stain reagent composition and a large molecule stainingcomposition.
 23. The method of claim 22, wherein the protective fluidlayer is an immiscible oil and wherein a density of the reagent dropletsis greater than a density of the immiscible oil.
 24. The method of claim22, wherein the large molecule staining composition comprises a largemolecule selected from the group consisting of an antibody, an antibodyconjugate, a multimer, and an enzyme; a surfactant; and a viscositymodifier, wherein the composition has a viscosity ranging from about 4cp to about 7 cp, and a surface tension ranging from about 20 dyne/cm toabout 40 dyne/cm.
 25. The method of claim 24, wherein the large moleculestaining composition is dispensed at a shear rate of less than about5×10⁵ s⁻¹.
 26. The method of claim 22, wherein the primary stain reagentcomposition comprises a dye, a surfactant, and a viscosity modifier,wherein the composition has a viscosity ranging from about 1 cp to about40 cp and a surface tension ranging from about 25 dyne/cm to about 45dyne/cm.
 27. The method of 26, wherein the primary stain reagentcomposition is dispensed at a shear rate of between about 1×10⁵ s⁻¹ andabout 1×10⁷ s⁻¹.